Analysis devices, kits, and related methods for digital quantification of nucleic acids and other analytes

ABSTRACT

Provided are devices and methods for effecting processing of samples, including essentially isothermal amplification of nucleic acids, at multiple reaction locations in a single device. In some embodiments, the disclosed devices and methods provide for effecting parallel sample processing in several hundred locations on a single device.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 13/440,371, filed Apr. 5,2012, which is a continuation-in-part application of application Ser.No. 13/257,811, filed Sep. 20, 2011; which is the National Stage ofInternational Application No. PCT/US2010/028316, filed on Mar. 23, 2010,which claims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication 61/262,375, filed on Nov. 18, 2009, and U.S. ProvisionalApplication No. 61/162,922, filed on Mar. 24, 2009, and U.S. ProvisionalApplication No. 61/340,872, filed on Mar. 22, 2010; this Applicationfurther claims the benefit of U.S. Provisional Application No.61/516,628, filed Apr. 5, 2011 and U.S. Provisional Application No.61/518,601, filed May 9, 2011; the content of all of which are herebyincorporated by reference in their entireties for any and all purposes.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under The United StatesGovernment has certain rights in this invention pursuant to Grant Nos. 1R01 EB012946, GM074961, and DP10D003584, awarded by the NationalInstitutes of Health (NIH); and Grant No. CHE-0526693, awarded by theNational Science Foundation. The United States Government has certainrights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 24, 2016, isnamed 33972US_CRF_sequencelisting.txt and is 4,172 bytes in size.

TECHNICAL FIELD

The present application relates to the field of microfluidics and to thefields of detection and amplification of biological entities.

BACKGROUND

Existing methods for nucleic acid amplification and quantitativeanalysis include real-time polymerase chain reaction (PCR) and real-timereverse-transcription polymerase chain reaction (RT-PCR). Real-timemethods are typically based on the detection of an exponential increaseof fluorescence intensity and rapid thermal cycling between thedissociation temperature (˜95° C.), annealing temperature (˜50° C.), andsynthesis temperature (˜70° C.).

Digital PCR is another method for quantitative analysis of nucleicacids. By dividing a diluted sample into a large number of small-volumereaction compartments, single copies of nucleic acid template can beconfined in isolated compartments and amplified by PCR. Only a “yes orno” readout is required, and the number of target molecules in thesample is determined by performing a statistical analysis on the numberof “positive” and “negative” wells. This method transfers theexponential amplification profile into a linear, digital format. Thesedigital PCR methods still require thermal cycling and accuratetemperature control, both of which may be challenging to ensure inresource-limited field conditions. Accordingly, there is a need in theart for, inter alia, devices and methods for isothermal processesapplicable to detection and even quantification of one or more analytes.The value of such devices and methods would be further enhanced if thedevices and methods were in at least some embodiments, manuallyportable.

SUMMARY

In meeting the described challenges, the present disclosure firstprovides methods, the methods comprising: effecting relative motionbetween a first substrate and a second substrate, the first substratehaving a first population of wells formed therein, the second substratehaving a second population of wells formed therein, the relative motionbetween the first and second substrates giving rise to at least somewells of the first population of wells being placed into fluidcommunication with at least some wells of the second population ofwells; and effecting contact between a first material disposed within atleast some of the first population of wells and a second materialdisposed within at least some of the second population of wells.

The present disclosure also provides methods, the methods comprisinginducing relative motion between a first substrate and a secondsubstrate so as to dispose a first material into first and secondpopulations of wells formed in at least one of the substrates; inducingrelative motion between the first and second substrates so as to disposea second material into third and fourth populations of wells formed atleast one of the substrates, the first and second materials beingcontacted to one another.

Further provided are devices. These devices (as well as those devicesdescribed in the priority documents) may be referred to as SlipChip™brand devices. In some embodiments, the device suitably comprising afirst substrate having a first population of wells formed therein, atleast one well of the first population of wells having at least onesatellite well disposed proximate to the at least one well, the at leastone satellite well being adapted to retain material from the at leastone well; a second substrate having a second plurality of wells formedtherein, the first and second substrates being slidably engageable withone another such that relative motion between the first and secondsubstrates places at least some of the first population of wells inregister with at least some of the second population of wells so as toform combined reaction chambers. The devices presented in the presentdisclosure may be of such a size that they are manually portable. Forexample, a device may define a cross-sectional dimension (e.g., height,width, thickness) that is in the range of 1 mm to about 1 cm, to about 5cm, to about 10 cm, or even to about 50 cm. The disclosed devices may belarger than the foregoing.

Additionally disclosed are kits. The disclosed kits suitably include afirst substrate having a first population of wells formed therein; asecond substrate having a second population of wells formed therein, thefirst and second substrates being superposable and slidably engageablewith one another such that relative motion between the substrates placesat least some of the first population of wells into fluid communicationwith at least some of the second population of wells; and a supply of atleast one reagent adapted to participate in amplification of nucleicacid.

Also provided are methods. The methods suitably include amplifying anucleic acid molecule, comprising contacting (a) a sample comprising atleast one nucleic acid molecule disposed at a plurality of first areas,with (b) at least one component of an amplification reagent disposed ina plurality of second areas, the contacting being effected by placingthe first and second areas into direct fluid communication with oneanother; and the contacting comprises effecting relative motion betweena substrate comprising the first area with a substrate comprising thesecond area; and exposing the area having the at least one nucleic acidmolecule to conditions effective for amplification of the at least onenucleic acid molecule.

The present disclosure also provides devices. The devices suitablyinclude a first substrate having a first population of areas, at leastone area of the first population of areas having at least one satellitearea disposed proximate to the at least one area, the at least onesatellite area being adapted to retain material from the at least onearea; a second substrate having a second plurality of area formedtherein, the first and second substrates being engageable with oneanother such that relative motion between the first and secondsubstrates places at least some of the first population of areas inregister with at least some of the second population of areas so as toplace the first and second areas into fluid communication with oneanother.

Additionally provided are methods of effecting amplification of at leastone nucleic acid target molecule. These methods suitably includecontacting (1) a sample material disposed in a plurality of first areas,the sample material comprising a nucleic acid target, and at least oneof the first areas containing one molecule of the nucleic acid target,with (2) a reactant material disposed in a plurality of second areas,the contacting being effected by pairwise placement of at least some ofthe first areas and at least some of the second areas into direct fluidcommunication with one another, the contacting effecting amplificationof at least one nucleic acid target molecule.

Further provided are methods, the methods suitably comprising dispersinga first sample that comprises at least one molecule of interest among aplurality of first areas, at least one of the first areas containing asingle molecule of interest; dispersing a reactant material into aplurality of second areas; and effecting pairwise placement of at leastsome of the plurality of first areas into direct fluid communicationwith at least some of the plurality of second areas so as to contactreactant material with the first sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the invention, there are shown in the drawingsexemplary embodiments of the invention; however, the invention is notlimited to the specific methods, compositions, and devices disclosed. Inaddition, the drawings are not necessarily drawn to scale. In thedrawings:

FIG. 1 illustrates RPA amplification of MRSA genomic DNA (5 pg/11 L) ina well plate at 25° C.;

FIGS. 2A-2N illustrate a schematic drawing of a two-step device fordigital RPA;

FIGS. 3A-3D illustrate fluorescence microphotographs and linescans ofRPA on a disclosed device before and after incubation at 39° C.

FIGS. 4A-4F illustrate digital RPA on a disclosed device with differentconcentration of MRSA gDNA;

FIG. 5 illustrates quantified results of digital RPA on a discloseddevice;

FIGS. 6A-6F illustrate a device for one-step digital RPA;

FIGS. 7A-7B illustrate comparative processes;

FIGS. 8A-8D illustrate a RPA two-step device for amplification of MRSAgDNA with incubation at different temperatures;

FIGS. 9A-9E illustrate food dye experiment demonstrated the operation ofslipping for a digital RPA device;

FIG. 10 illustrate a “streaky” distribution of positive wells wasobtained when RPA was pre-initiated off-chip for one minute and loadedonto the chip via pipetting over 4 minutes;

FIGS. 11A-11E illustrate a schematic drawing showing procedures toperform digital PCR by using the two-step device;

FIG. 12 illustrate experimental results showing digitalreverse-transcription polymerase chain reaction (RT-PCR) and digitalNASBA performed on a disclosed device using the same template andinitial concentration, showing parallel results at three differentconcentrations;

FIG. 13 illustrates NASBA enzymes (reverse transcriptase [RT] and RNaseH) conversion of RNA template into cDNA that is then used to create manycopies of antisense RNA by T7 polymerase—antisense RNA is then used togenerate more cDNA which makes even more antisense RNA, and theantisense RNA product can hybridize to a beacon leading to generation ofa strong fluorescent signal, or it could be hybridized to other speciesto generate a vsual readout;

FIG. 14 illustrates a schematic of an exemplary two-stage device design.The design includes 1280 of each well type; the filled wells are about2.6 nL in volume for the glass chips and about 3 nL in volume forplastic chips. Thermal expansion wells are about 0.3 nL in volume;

FIG. 15 is a table summary of beacon design and signal increase to theNASBA product of HIV;

FIGS. 16A-16B illustrate an example of digital NASBA of HIV—FIG. 16Afluorescent image of an exemplary device, and FIG. 16B linescan of wells(dashed line, within white box) showing approximately 20 fold increasein signal using beacon design V3;

FIG. 17 illustrates a comparison of digital RT-PCR and digital NASBAshowing good agreement between results from experiments were performedusing on chip initiation;

FIGS. 18A-18C illustrate testing viability of loading premixed NASBA atseveral pre-incubation temperatures—FIG. 18A time course experiments onice (square), at room temperature (circle) and at 30° C. (triangle).Images of NASBA results at 30° C., FIG. 18B immediately after mixing,and FIG. 18C after about 30 minutes of pre-incubation;

FIGS. 19A-19C illustrate optimizing silver amplification in wells andpreliminary results in a disclosed device. FIG. 19A: Rapid reaction rateand sensitivity to AuNP concentration, with clean background foroptimized silver amplification conditions, FIG. 19B: Comparing effect ofPEGThiol and demonstration of signal generation from complete magneticbead:analyte:AuNP complex, FIG. 19C: Demonstration of clean backgroundand visual signal of AuNP at low (5 pM) concentration in the device; and

FIGS. 20A-20B illustrate a single molecule Immuno-PCR using PSA astarget protein, showing FIG. 20A an expanded view of a section of thedevice showing digital readout of PCR and distribution of beads. Onebright spot (larger spot) stands for one amplified reaction while onedark spot (smaller spot) stands for one magnetic bead, and FIG. 20Bfraction of positive wells with beads (signal) and without beads(background).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingfigures and examples, which form a part of this disclosure. It is to beunderstood that this invention is not limited to the specific devices,methods, applications, conditions or parameters described and/or shownherein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed invention. Also, as used in thespecification including the appended claims, the singular forms a, an,and the include the plural, and reference to a particular numericalvalue includes at least that particular value, unless the contextclearly dictates otherwise.

The term plurality as used herein, means more than one. When a range ofvalues is expressed, another embodiment includes from the one particularvalue and/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent about, it will beunderstood that the particular value forms another embodiment. Allranges are inclusive and combinable. Any documents cited herein areincorporated herein by reference in their entireties for any and allpurposes.

Certain description herein refers to “areas.” It should be understoodthat the term “area” refers to a site where two or more materials may becontacted with one another. The term may also refer to a region thatmaintains a material thereon, therealong, or therein. An “area” may takeon a physical structure such as a hole, well, cavity, or indentation,and may also have any cross-sectional shape along its length, width, ordepth, such as rectangular, circular, or triangular. An area may also bea region of a substrate, which region may include a treatment to renderit hydrophilic or hydrophobic.

For convenience and also for purposes of ease of illustration, a numberof exemplary embodiments provided herein describe areas by illustratingareas with well structures. Such description and illustration should notbe taken as limiting the scope of the present disclosure to embodimentsthat feature wells, as the disclosed devices and methods may be appliedto any one or more of the various types of areas described above. Theterm “wells” should be understood as being representative of “areas,”and that other types of areas may be used in place of the “wells” usedto illustrate an exemplary embodiment.

In a first aspect, the present disclosure provides methods. The methodssuitably include effecting relative motion between a first substrate anda second substrate. The first substrate suitably has a first populationof wells formed therein, and the second substrate suitably has a secondpopulation of wells formed therein.

It should be understood that a substrate may have multiple populationsof areas (e.g., wells) formed therein. As one example, the firstsubstrate may include one population of wells that are placed into fluidcommunication with one another by way of a first conduit formed in thesubstrate, the conduit being configured to allow filling of the wellsfrom a source exterior to the substrate (e.g., FIG. 2). The firstsubstrate may include another population of wells that is not in fluidcommunication with the first population of wells. This other populationof wells may be placed into fluid communication with one another by wayof a conduit formed in the substrate, or the wells may be formed in thesubstrate without connection to the environment exterior to thesubstrate.

The first and second substrate may be configured such that relativemotion between the first and second substrates gives rise to at leastsome wells of the first population of wells being placed into fluidcommunication with at least some wells of the second population ofwells. This relative motion may be referred to in some places forconvenience as “slipping,” and may refer to linear motion (as shown inexemplary FIG. 2), but may also to rotational motion or other non-linearmovement. The relative motion may be effected manually, or by mechanicalor other automated means.

The methods also suitably include effecting contact between a firstmaterial disposed within at least some of the first population of wellsand a second material disposed within at least some of the secondpopulation of wells. This contact may be effected, for example, byplacing a well of the first population into fluid communication with awell of the second population, as shown in exemplary FIG. 2, whereplacing two wells into fluid communication with one another effectscontact between the two fluids. It should be understood that materialsdisposed within the wells are not limited to liquids, as solid materials(e.g., dried reagents) and even gases may be disposed within the wellsof the disclosed devices.

In some embodiments, the user may introduce the first material into apopulation of wells by exerting the one or more materials into a conduitformed in the second substrate, the conduit being in fluid communicationwith the first population of wells. This is illustrated in exemplaryFIG. 2 and FIG. 6, which show introduction of a material via a conduitinto a population of wells of a substrate. Similarly, a user mayintroduce the second material into the second population of wells byexerting the one or more materials into a conduit formed in the secondsubstrate, the conduit being in fluid communication with the secondpopulation of wells. A user may also dispose a material into a well bydripping, pipetting, vapor deposition, and the like; introduction ofmaterial into a well is not limited to doing so by way of conduits. Auser may use a device (pipette, syringe, and the like) that is adaptedto engage with a conduit formed in the device.

In some embodiments, at least some of the first material remainsuncontacted with the second material. Some of the second material mayalso remain uncontacted with the first material. This may be done tocreate control wells for a given experiment or analysis.

The methods may also include amplifying one or more nucleic acidspresent within the first population of wells, the second population ofwells, or both. A variety of amplification techniques are known in thefield; amplification techniques that are performed essentiallyisothermally are considered especially suitable. By isothermal is meanta reaction that involves fewer than 10 changes in temperature. It shouldbe understood that although isothermal techniques are particularlyuseful, the present disclosure is not limited to isothermalamplification. A non-exclusive listing of suitable amplificationtechniques includes loop-mediated amplification, nucleic acid sequencebased amplification, recombinase polymerase amplification, rollingcircle amplification, helicase-dependent amplification,transcription-mediated amplification, multiple displacementamplification, strand-displacement amplification, and the like. Anexemplary listing of amplification techniques is provided in applicationPCT/US2010/028316, the entirety of which is incorporated herein byreference. Combinations of techniques may be used. For example, one setof wells may be used in the amplification of nucleic acids by a firsttechnique, and a separate set of wells maybe used in the amplificationof nucleic acids by a second technique.

A non-exclusive listing of suitable isothermal amplification techniquesare provided below. These techniques are illustrative only, and do notlimit the present disclosure.

A first set of suitable isothermal amplification technologies includesNASBA, and RT-RPA. These amplification techniques can operate at 40 deg.C0 (a lower temperature preferred for certain POC devices): NASBA(product: RNA), RT-RPA (product: DNA), RT-LAMP using one of LAMP HIV-RNA6-primer sets, transcription-mediated amplification (TMA, 41 deg. C.),helicase dependent amplification (HAD, 65 deg. C.), andstrand-displacement amplification (SDA, 37 deg. C.),

In addition to standard PCR techniques, the disclosed methods anddevices are compatible with isothermal amplification techniques such asloop-mediated amplification (LAMP), Recombinase polymerase amplification(RPA), nucleic acid sequence based amplification (NASBA),transcription-mediated amplification (TMA), helicase-dependentamplification (HAD), rolling circle amplification (RCA), andstrand-displacement amplification (SDA). The multivolume SlipChip can beused to digitize such platforms.

Other isothermal amplification methods are also suitable. Isothermalexponential amplification reaction (EXPAR) can amplify a 10-20 bptrigger oligonucleotide generated from a genomic target more than 106times in less than 10 minutes at 55 deg. C. by repeating cycles ofpolymerase and endonuclease activity, and has been coupled withDNA-functionalized gold nanospheres for the detection of herpes simplexvirus. Isothermal and chimeric primer-initiated amplification of nucleicacids (ICANs) amplify target DNA at 55 deg. C. using a pair of50-DNA-RNA-30 primers and the activity of RNase H and strand displacingpolymerase.

Signal-mediated amplification of RNA technology (SMART) produces copiesof an RNA signal at 41 deg. C. in the presence of an RNA or DNA targetby way of the three-way junction formed between the target and twoprobes, one of which contains the RNA signal sequence and a T7 promotersequence for T7 RNA polymerase. The single stranded RNA product may bedetected by hybridization-based methods and because the signal isindependent of the target, SMART can be easily adapted for detection ofdifferent target sequences. Cyclic enzymatic amplification method (CEAM)detects nucleic acids in the picomolar range in less than 20 minutes at37 deg. C. using a displacing probe and Exonuclease III (Exo III) togenerate amplification of fluorescent signal in the presence of atarget. Isothermal target and signaling probe amplification (iTPA)combines the principle of ICAN and the inner-outer probe concept of LAMPalong with fluorescence resonance energy transfer cycling probetechnology (FRET CPT) for simultaneous target and signal amplificationin 90 minutes at 60 deg. C., and has been shown to detect Chlamydiatrachomatis at single copy level.

Other suitable amplification methods include ligase chain reaction(LCR); amplification methods based on the use of Q-beta replicase ortemplate-dependent polymerase; helicase-dependent isothermalamplification; strand displacement amplification (SDA); thermophilic SDAnucleic acid sequence based amplification (3SR or NASBA) andtranscription-associated amplification (TAA).

Non-limiting examples of PCR amplification methods include standard PCR,AFLP-PCR, Allele-specific PCR, Alu-PCR, Asymmetric PCR, BiasedAllele-Specific (BAS) Amplification, Colony PCR, Hot start PCR, InversePCR (IPCR), In situ PCR (ISH), Intersequence-specific PCR (ISSR-PCR),Long PCR, Multiplex PCR, Nested PCR, Quantitative PCR, ReverseTranscription PCR (RT-PCR), Real Time PCR, Single cell PCR, Solid phasePCR, Universal Size-Specific (USS-PCR), branched-DNA technology, and thelike

A variety of specific amplification techniques are described below. Eachof these techniques is suitably performed by the disclosed devices andmethods. Allele-specific PCR is a diagnostic or cloning technique basedon single-nucleotide polymorphisms (SNPs) (single-base differences inDNA). It requires some knowledge of a DNA sequence, includingdifferences between alleles, and uses primers whose 3′ ends encompassthe SNP. PCR amplification may be less efficient in the presence of amismatch between template and primer, so successful amplification withan SNP-specific primer signals presence of the specific SNP in asequence.

Assembly PCR or Polymerase Cycling Assembly (PCA) is an artificialsynthesis of long DNA sequences by performing PCR on a pool of longoligonucleotides with short overlapping segments. The oligonucleotidesalternate between sense and antisense directions, and the overlappingsegments determine the order of the PCR fragments, thereby selectivelyproducing the final long DNA product.

Asymmetric PCR preferentially amplifies one DNA strand in adouble-stranded DNA template. It is used in sequencing and hybridizationprobing where amplification of only one of the two complementary strandsis required. PCR is carried out as usual, but with a great excess of theprimer for the strand targeted for amplification. Because of the slow(arithmetic) amplification later in the reaction after the limitingprimer has been used up, extra cycles of PCR are required. A recentmodification on this process, known as Linear-After-The-Exponential-PCR(LATE-PCR), uses a limiting primer with a higher melting temperature(Tm) than the excess primer to maintain reaction efficiency as thelimiting primer concentration decreases mid-reaction.

Helicase-dependent amplification is similar to traditional PCR, but usesa constant temperature rather than cycling through denaturation andannealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, isused in place of thermal denaturation.

Hot start PCR is a technique that reduces non-specific amplificationduring the initial set up stages of the PCR. It may be performedmanually by heating the reaction components to the denaturationtemperature (e.g., 95° C.) before adding the polymerase. Specializedenzyme systems have been developed that inhibit the polymerase'sactivity at ambient temperature, either by the binding of an antibody orby the presence of covalently bound inhibitors that dissociate onlyafter a high-temperature activation step. Hot-start/cold-finish PCR isachieved with new hybrid polymerases that are inactive at ambienttemperature and are instantly activated at elongation temperature.

Intersequence-specific PCR (ISSR) is a PCR method for DNA fingerprintingthat amplifies regions between simple sequence repeats to produce aunique fingerprint of amplified fragment lengths.

Inverse PCR is commonly used to identify the flanking sequences aroundgenomic inserts. It involves a series of DNA digestions and selfligation, resulting in known sequences at either end of the unknownsequence.

Ligation-mediated PCR: uses small DNA linkers ligated to the DNA ofinterest and multiple primers annealing to the DNA linkers; it has beenused for DNA sequencing, genome walking, and DNA footprinting.

Methylation-specific PCR (MSP) is used to detect methylation of CpGislands in genomic DNA. DNA is first treated with sodium bisulfate,which converts unmethylated cytosine bases to uracil, which isrecognized by PCR primers as thymine. Two PCRs are then carried out onthe modified DNA, using primer sets identical except at any CpG islandswithin the primer sequences. At these points, one primer set recognizesDNA with cytosines to amplify methylated DNA, and one set recognizes DNAwith uracil or thymine to amplify unmethylated DNA. MSP using qPCR canalso be performed to obtain quantitative rather than qualitativeinformation about methylation.

Miniprimer PCR uses a thermostable polymerase (S-Tbr) that can extendfrom short primers (“smalligos”) as short as 9 or 10 nucleotides. Thismethod permits PCR targeting to smaller primer binding regions, and isused to amplify conserved DNA sequences, such as the 16S (or eukaryotic18S) rRNA gene.

Multiplex Ligation-dependent Probe Amplification (MLPA) permits multipletargets to be amplified with only a single primer pair, as distinct frommultiplex-PCR.

Multiplex-PCR: consists of multiple primer sets within a single PCRmixture to produce amplicons of varying sizes that are specific todifferent DNA sequences. By targeting multiple genes at once, additionalinformation may be gained from a single test-run that otherwise wouldrequire several times the reagents and more time to perform. Annealingtemperatures for each of the primer sets must be optimized to workcorrectly within a single reaction, and amplicon sizes. That is, theirbase pair length should be different enough to form distinct bands whenvisualized by gel electrophoresis.

Nested PCR: increases the specificity of DNA amplification, by reducingbackground due to non-specific amplification of DNA. Two sets of primersare used in two successive PCRs. In the first reaction, one pair ofprimers is used to generate DNA products, which besides the intendedtarget, may still consist of non-specifically amplified DNA fragments.The product(s) are then used in a second PCR with a set of primers whosebinding sites are completely or partially different from and located 3′of each of the primers used in the first reaction. Nested PCR is oftenmore successful in specifically amplifying long DNA fragments thanconventional PCR, but it requires more detailed knowledge of the targetsequences.

Overlap-extension PCR or Splicing by overlap extension (SOE): a geneticengineering technique that is used to splice together two or more DNAfragments that contain complementary sequences. It is used to join DNApieces containing genes, regulatory sequences, or mutations; thetechnique enables creation of specific and long DNA constructs.

Quantitative PCR (Q-PCR): used to measure the quantity of a PCR product(commonly in real-time). It quantitatively measures starting amounts ofDNA, cDNA, or RNA. Q-PCR is commonly used to determine whether a DNAsequence is present in a sample and the number of its copies in thesample. Quantitative real-time PCR can have a high degree of precision.QRT-PCR (or QF-PCR) methods use fluorescent dyes, such as Sybr Green,EvaGreen or fluorophore-containing DNA probes, such as TaqMan, tomeasure the amount of amplified product in real time. It is alsosometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR orRTQ-PCR are more appropriate contractions, since RT-PCR commonly refersto reverse transcription PCR (see below), often used in conjunction withQ-PCR.

Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reversetranscriptase reverse transcribes RNA into cDNA, which is then amplifiedby PCR. RT-PCR is widely used in expression profiling, to determine theexpression of a gene or to identify the sequence of an RNA transcript,including transcription start and termination sites. If the genomic DNAsequence of a gene is known, RT-PCR can be used to map the location ofexons and introns in the gene. The 5′ end of a gene (corresponding tothe transcription start site) is typically identified by RACE-PCR (RapidAmplification of cDNA Ends).

Solid Phase PCR: encompasses multiple meanings, including PolonyAmplification (where PCR colonies are derived in a gel matrix, forexample), Bridge PCR[32] (primers are covalently linked to asolid-support surface), conventional Solid Phase PCR (where AsymmetricPCR is applied in the presence of solid support bearing primer withsequence matching one of the aqueous primers) and Enhanced Solid PhasePCR (where conventional Solid Phase PCR can be improved by employinghigh Tm and nested solid support primer with optional application of athermal ‘step’ to favour solid support priming).

Thermal asymmetric interlaced PCR (TAIL-PCR) may be useful for isolationof an unknown sequence flanking a known sequence. Within the knownsequence, TAIL-PCR uses a nested pair of primers with differingannealing temperatures; a degenerate primer is used to amplify in theother direction from the unknown sequence.

Touchdown PCR (Step-down PCR) is a variant of PCR that aims to reducenonspecific background by gradually lowering the annealing temperatureas PCR cycling progresses. The annealing temperature at the initialcycles is usually a few degrees (3-5° C.) above the Tm of the primersused, while at the later cycles, it is a few degrees (3-5° C.) below theprimer Tm. The higher temperatures give greater specificity for primerbinding, and the lower temperatures permit more efficient amplificationfrom the specific products formed during the initial cycles.

PAN-AC uses isothermal conditions for amplification, and may be used inliving cells.

Universal Fast Walking is useful for genome walking and geneticfingerprinting using a more specific ‘two-sided’ PCR than conventional‘one-sided’ approaches (using only one gene-specific primer and onegeneral primer—which can lead to artefactual ‘noise’) by virtue of amechanism involving lariat structure formation. Streamlined derivativesof UFW are LaNe RAGE (lariat-dependent nested PCR for rapidamplification of genomic DNA ends), 5′RACE LaNe, and 3′RACE LaNe.

COLD-PCR (co-amplification at lower denaturation temperature-PCR) is amodified Polymerase Chain Reaction (PCR) protocol that enriches variantalleles from a mixture of wildtype and mutation-containing DNA.

An alternative isothermal amplification and detection method that isisothermal in nature is described athttp://www.invaderchemistry.com/(Invader Chemistry). This method may beperformed by the disclosed devices and methods. Another alternativeamplification technique (so-called qPCR) is disclosed by MNAzyme(http://www.speedx.com.au/MNAzymeqPCR.html), which technique is alsosuitable for the presently disclosed devices and methods.

One may also effect amplification based on nucleic acid circuits (whichcircuits may be enzyme-free). The following references (all of which areincorporated herein by reference in their entireties) describe exemplarycircuits; all of the following are suitable for use in the discloseddevices and methods: Li et al., “Rational, modular adaptation ofenzyme-free DNA circuits to multiple detection methods,” Nucl. AcidsRes. (2011) doi: 10.1093/nar/gkr504; Seelig et al., “Enzyme-Free NucleicAcid Logic Circuits,” Science (Dec. 8, 2006), 1585-1588; Genot et al,“Remote Toehold: A Mechanism for Flexible Control of DNA HybridizationKinetics,” JACS 2011, 133 (7), pp 2177-2182; Choi et al., “Programmablein situ amplification for multiplexed imaging of mRNA expression,”Nature Biotechnol, 28:1208-1212, 2010; Benner, Steven A., and A. MichaelSismour. “Synthetic Biology.” Nat Rev Genet 6, no. 7 (2005): 533-543;Dirks, R. M., and N. A. Pierce. “Triggered Amplification byHybridization Chain Reaction.” Proceedings of the National Academy ofSciences of the United States of America 101, no. 43 (2004): 15275;Graugnard, E., A. Cox, J. Lee, C. Jorcyk, B. Yurke, and W. L. Hughes.“Kinetics of DNA and Rna Hybridization in Serum and Serum-Sds.”Nanotechnology, IEEE Transactions on 9, no. 5 (2010): 603-609; Li,Bingling, Andrew D. Ellington, and Xi Chen. “Rational, ModularAdaptation of Enzyme-Free DNA Circuits to Multiple Detection Methods.”Nucleic Acids Research, (2011); Li, Q., G. Luan, Q. Guo, and J. Liang.“A New Class of Homogeneous Nucleic Acid Probes Based on SpecificDisplacement Hybridization.” Nucleic Acids Research 30, no. 2 (2002):e5-e5; Picuri, J. M., B. M. Frezza, and M. R. Ghadiri. “UniversalTranslators for Nucleic Acid Diagnosis.” Journal of the AmericanChemical Society 131, no. 26 (2009): 9368-9377; Qian, Lulu, and ErikWinfree. “Scaling up Digital Circuit Computation with DNA StrandDisplacement Cascades.” Science 332, no. 6034 (2011): 1196-1201;Tsongalis, G. J. “Branched DNA Technology in Molecular Diagnostics.”American journal of clinical pathology 126, no. 3 (2006): 448-453; VanNess, Jeffrey, Lori K. Van Ness, and David J. Galas. “IsothermalReactions for the Amplification of Oligonucleotides.” Proceedings of theNational Academy of Sciences 100, no. 8 (2003): 4504-4509; Yin, Peng,Harry M. T. Choi, Colby R. Calvert, and Niles A. Pierce. “ProgrammingBiomolecular Self-Assembly Pathways.” Nature 451, no. 7176 (2008):318-322; Zhang, D. Y., and E. Winfree. “Control of DNA StrandDisplacement Kinetics Using Toehold Exchange.” Journal of the AmericanChemical Society 131, no. 47 (2009): 17303-17314; Zhang, David Yu,Andrew J. Turberfield, Bernard Yurke, and Erik Winfree. “EngineeringEntropy-Driven Reactions and Networks Catalyzed by DNA.” Science 318,no. 5853 (2007): 1121-1125; Zhang, Z., D. Zeng, H. Ma, G. Feng, J. Hu,L. He, C. Li, and C. Fan. “A DNA-Origami Chip Platform for Label-FreeSNP Genotyping Using Toehold-Mediated Strand Displacement.” Small 6, no.17 (2010): 1854-1858.

In some embodiments, the one or more nucleic acids may reside on a labelbound to a protein. This may be applied in immuno-amplificationtechniques, as described elsewhere herein, which techniques enabledetection and quantification of proteins.

In certain embodiments of the disclosed technology, at least one well ofthe first population of wells, the second population of wells, or both,is disposed proximate to a satellite well. Such a satellite well issuitably adapted to retain material from the at least one well. This isillustrated in, e.g., non-limiting FIG. 2 and FIG. 6, which show showssatellite wells disposed proximate to the Type II wells shown in thefigure. The satellite wells may be used to retain material that may exitanother well, e.g., as a result of thermal expansion.

The relative motion between the first and second substrates places atleast some of the first population of wells in register with at leastsome of the second population of wells so as to form combined reactionchambers. This are shown in illustrative FIG. 2 and FIG. 6, which showformation of such reaction chambers resulting from the registry betweenType I and Type II wells. The relative motion may give rise to 1, 10,100, 1000, 10,000, or even more such reaction chambers on a substrate oreven within a device. As one example, the relative motion between twosubstrates may pairwise place about 10 first areas (e.g., wells) intodirect fluid communication (e.g., by placing into register) with 10second areas. The relative motion may pairwise place about 50, 100, oreven 1000 first areas into direct fluid communication with 50, 100, oreven 1000, respectively, second areas.

Wells formed on a substrate may have comparatively small volumes, e.g.,about 0.1, 1, 5, 10, 50, 100, or even about 1000 nL per well. Asubstrate may include wells of two or more volumes, and given, discretepopulation of wells (e.g., a set of wells that are in fluidcommunication with a conduit formed in the substrate in which the wellsreside) may include wells of two or more volumes.

In other embodiments, the present disclosure provides methods, themethods including inducing relative motion between a first substrate anda second substrate so as to dispose a first material into first andsecond populations of wells formed in at least one of the substrates;inducing relative motion between the first and second substrates so asto dispose a second material into third and fourth populations of wellsformed at least one of the substrates, the first and second materialsbeing contacted to one another.

In some embodiments, the user may introduce the first material to atleast one well by exerting the material through a conduit in fluidcommunication with the well. The user may also introduce the secondmaterial to at least one well by exerting the material through a conduitin fluid communication with the well.

As described elsewhere herein, at least some of the first material mayremain uncombined with the second material. Likewise, at least some ofthe second material may remain uncombined with the first material. Thisis shown in FIG. 2, which figure shows control wells that do not containmixed materials.

The user may also amplify one or more nucleic acids present within apopulation of wells. Such amplification may be effected with temperaturecycling; isothermal methods of amplification are considered especiallysuitable. Suitable methods of amplification are described elsewhereherein. Suitable well and substrate configurations—including satellitewells—are described elsewhere herein.

It should be understood that the present disclosure is not limited toisothermal processes, and that the disclosed devices and methods may beadapted to effect other molecular reactions, including single-moleculereactions. For example, a sample may be introduced to a first set ofcompartments such that at least one well contains (or is estimated tocontain) only a single molecule of sample. The sample may be undergo areaction (e.g., labeling, neutralizing, acidification, digestion,ligation, translation, transcription, reverse transcription,crystallization, incubation, dissolution, detection and the like) inthat first set of compartments. With specific regard to detectionreactions, the detection reaction may include molecular beacons,sequencing, enzymatic reactions, fluorogenic reactions, colorimetricreactions, and the like. The detection methods provided in priorityapplication PCT/US2010/028316 (incorporated herein in its entirety) aresuitable for the disclosed devices and methods.

The user may effect relative motion between that first set ofcompartments and a second set of compartments that contains a particularreagent so as to place the first and second sets of compartments intoregister and direct fluid communication with one another. The material(i.e., reacted sample) in the first set of wells then contacts thereagent in the second set of wells, and the reacted sample may thenreact with the reagent in the second set of wells. In some embodiments,this reaction may be an amplification reaction, or labeling,neutralizing, acidification, digestion, ligation, translation,transcription, reverse transcription, crystallization, incubation,dissolution, and the like. The reaction may also be a detectionreaction. Accordingly, as described above, a given sample that residesin a first set of compartments may undergo multiple, sequentialreactions as the sample is exposed to other reagents by way of relativemotion between the compartment(s) in which the sample is disposed andother compartments that contain other reagents. This in turn enableseffecting sequential processing on multiple sample compartments inparallel.

Some embodiments of the devices and methods disclosed here provide foramplification of nucleic acids, followed by recovery of the amplifiedmaterial. Such recovery may be carried out, in some embodiments, byaccessing individual wells of a device. In some embodiments, recoverymay be achieved by combining material from multiple wells, for exampleby slipping a device to the loading position and using a carrier fluidto expel the material from the device. Such recovery may be used foradditional analysis of nucleic acids, such as sequencing, genotyping,analysis of methylation patterns, and identification of epigeneticmarkers. In some embodiments, recovered material may be removed from thedevice. In some embodiment, recovered material may be transferred toanother device, or another region of the same device. Amplification maybe carried out by the methods described herein or by other methods knownin the art or by their combinations.

Some embodiments of the devices and methods disclosed here provide for amethod of carrying out sequential reactions on multiple compartments.For example, a first fluid containing one or more molecules of interestcan be introduced into a device. Compartmentalization of the fluid maybe carried out by slipping to create the first set of compartments. Insome preferred embodiments, some of the first compartments have singlemolecules of interest. A first reaction can be carried out, for example,by incubation at a particular temperature or combination oftemperatures, by applying fields and gradients, and/or by other means.Second compartments of a second fluid, containing a reagent, can becreated either in parallel with creating the first compartments, orprior to first compartments, or subsequently to the first compartments.After the desired extent of the first reaction, the first compartmentscontaining the first reaction mixtures can be combined with the secondcompartments to carry out the second reaction. The second reaction couldbe carrying out on the product of the first reaction (for example, asecond detection reaction on the product of the first nucleic acidamplification reaction), or could be performed on other components ofthe first reaction mixture (for example detecting a protein in thesecond reaction following a detection of a nucleic acid in the firstreaction). In some embodiments, such sequential reactions can be carriedout in parallel, for example in devices such as those illustrated inFIG. 2 and FIG. 14. In the example given in FIG. 14, the first reactioncan be carried out, for example, during the stage labeled “isolatecomponent 1” and the second reaction can be carried out during the stagelabeled “react and readout”.

As an additional example, molecules of interest can be molecules ofnucleic acids. The first reaction could be an amplification reaction,and the second reaction could be a subsequent amplification reaction.The first reaction can be a reverse-transcription reaction and thesecond reaction could be an amplification reaction. The first reactioncan be an amplification reaction, and the second reaction can be adetection reaction. The first reaction could be a sequencing librarypreparation reaction, and the second reaction could be an amplificationreaction. Examples of other first and second reactions, which should notbe considered limiting examples, include detection reactions. Anon-limiting listing of reactions is provided at, e.g., paragraphs0112-0124 in published United States Application 2011/0166044, theentirety of which is incorporated herein by reference. The exemplaryembodiments disclosed herein regarding NASBA and LAMP amplificationprocesses illustrate the foregoing, namely, a multistep reactionprocess. Additional examples of embodiments that feature first andsecond reactions are described in priority application PCT/US2010/028316at, e.g., at paragraph 0194 and elsewhere in that application. In oneembodiment, the first reaction may be nucleic acid amplification (e.g.,PCR), and the second reaction may be a recovery of the amplificationproducts. In some embodiments, recovery of amplification products neednot necessarily be performed by a reaction. The amplification productsmay be recovered by a filter, a monolith, and the like—materials thatpreferentially adsorb amplification products are considered especiallysuitable.

Recovery of products (e.g., amplified nucleic acid products) may beeffected by removing the product (e.g., by pipette, syringe, or otherdevice). Recovery may also be effected by actuating amplificationproduct fluid with a carrier or lubricating fluid, as described inpriority application PCT/US2010/028316. Further methods of moving fluidsare also described in priority application PCT/US2010/028316.

As one example, a user might combine nucleic acids and amplification toform an admixture. The user may then introduce that admixture into adevice according to the present disclosure so as to distribute theadmixture among multiple areas (e.g., wells). The areas may be in fluidcommunication with one another, but may also be in fluid isolation fromone another. Once the admixture is disposed into these areas, the usermay apply conditions (heat, light) sufficient to effect a reaction inthe admixture. The reacted admixture may then be distributed among othercompartments, where the reacted admixture undergoes further reactions.

It should be understood that the devices and methods disclosed hereinmay be configured to effect a time delay between steps. For example, asample and reagent may be introduced into a set of reactioncompartments. At that time, the user may apply heat to the materials soas to effect a reaction between the sample and reagent. The user maythen, after a time sufficient to allow the reaction to reach a desiredstage, effect contact between the reacted material and another reagent,as described elsewhere herein. Delay may be effected manually or byaction of a controller that actuates substrates, valves, pumps, or othercomponents of the disclosed technology. Delays in the range of from onethousandth of a second to one second are suitable delay lengths, as suchdelays may be sufficient to allow sufficient progress of a reaction.Thus, in some embodiments, the devices may effect a first reactionduring a delay, place the reaction products into contact with anadditional reagent, and then effect a delay so as to allow a secondreaction to take place.

This may be illustrated by reference to FIG. 6. In that figure, a usermay (panel B) add first and second solutions to separate sets of wells.The first solution may be a solution (e.g., nucleic acid andamplification reagent) that undergoes a reaction in place (e.g., byapplication of heat, light, or by passage of time). The separate sets ofwells may be combined so as to effect contact between the reacted firstsolution in the first set of wells and the second solution in a secondset of wells.

It should also be understood that the present disclosure is not limitedto application to molecules, as the disclosed devices and methods may beapplied to organisms (such as those described in paragraph 0133 ofpriority application PCT/US2010/028316 and also elsewhere in thatapplication), single cells, single biological particles (e.g.,bacteria), single vesicles, single exosomes, single viruses, singlespores, lipoprotein particles, and the like, and single non-biologicalparticles. Furthermore, it should also be understood that the discloseddevices and methods may be applied to stochastic confinement (describedin, for example, “Stochastic Confinement to Detect, Manipulate, AndUtilize Molecules and Organisms,” application no. PCT/US2008/071374),and reactions and manipulations of stochastically confined objects. Asone non-limiting example, biological samples may be assessed for thepresence or level of certain bacteria, such as those organisms thatserve as markers for bacterial vaginosis. This assessment may beperformed by amplifying nucleic acids that may be present in the sampleand correlating the levels of those nucleic acids to the presence orabsence of the marker organisms. An exemplary approach to such ananalysis is found at http://www.viromed.com/client/cats/BV %20LAB.pdf.

Also presented are devices. The devices suitably include a firstsubstrate having a first population of wells formed therein, at leastone well of the first population of wells having at least one satellitewell disposed proximate to the at least one well, the at least onesatellite well being adapted to retain material from the at least onewell; a second substrate having a second plurality of wells formedtherein, the first and second substrates being slidably engageable withone another such that relative motion between the first and secondsubstrates places at least some of the first population of wells inregister with at least some of the second population of wells so as toform combined reaction chambers.

Exemplary devices are shown and described in FIGS. 2 and 6. At least oneof the substrates may have a thickness in the range of from about 10micrometers to about 500, about 1000, or even about 2000 micrometers.Substrates having a thickness in the range of from about 200 micrometersto about 700 micrometers are considered especially suitable. A well mayhave a volume in the range of from about 1 nL to about 5, 10, 25, 50,100, 250, 500, or even about 1000 nL; a well need not be fully filled inorder for the devices (and methods) disclosed herein to operate.

The relative motion (which may be referred to as actuating) may giverise to at least one reaction vessel defined by a well of the firstpopulation of wells in fluid communication with a well of the secondpopulation of wells, the at least one reaction vessel. The reactionvessel may be in fluidic isolation from other wells or other reactionvessels, also fluidic isolation is not q requirement. The reactionvessel may have a volume in the range of from about 1 nL to about 5, 10,25, 50, 100, 250, 500, or even about 2000 or even 5000 nL, depending onthe user's needs. Exemplary fabrication methods for such devices are setforth in Du et al., Lab Chip 2009, 9, 2286-2292. A population of wellsof the disclosed devices may include two or more wells of differentvolumes. As described elsewhere herein, a reaction vessel may include atleast one satellite well, which wells are described elsewhere herein,and are shown in exemplary FIGS. 2 and 6.

It should be understood that the methods may include one, two, or moreapplications of relative motion between substrates. For example, a firstrelative motion (e.g., rotation) may be applied so as to place first andsecond sets of wells into fluid communication with one another. Once thecontents of the first and second wells have contacted one another,additional rotation may be applied to place the wells with mixedcontents into fluid communication with another set of wells withdifferent contents, which in turns enables the user to effect processesthat require separate and/or sequential mixing steps of two, three, ormore sample volumes. This might be done, for example, to (1) mixmaterials in well A and well B in well A; and then (2) to contact themixed materials in well A with a buffer in well C so as to dilute thecontents of well A. Alternatively, the mixed contents of well A mightthen be contacted (via relative motion of substrates) with well C suchthat the contents of well C may react with the contents of well A (whichwell included the contents of well A and well B).

The devices may be configured such that relative motion between thesubstrates gives rise to at least about 2, 5, 10, 100, 500, 1000, 2500,or even about 10,000 (including any and all intermediate values)combined reaction chambers. The disclosed devices may include one ormore supplies of a reagent or reagents that are adapted (or selected) toparticipate in nucleic acid amplification. Such reagents may be packagedtogether with the first and second substrates. A partial, non-exclusivelisting of such reagents includes buffers, primers, and the like. Thereagent may be dried and disposed in a well before or after thesubstrates are engaged with one another. The reagent may also bedisposed within a well and then dried.

The present disclosure also provides kits. The kits suitably include afirst substrate having a first population of wells formed therein; asecond substrate having a second population of wells formed therein, thefirst and second substrates being superposable and slidably engageablewith one another such that relative motion between the substrates placesat least some of the first population of wells into fluid communicationwith at least some of the second population of wells; and a supply of atleast one reagent adapted to participate in amplification of nucleicacid. The kits may, however, include reagents in addition to or even inplace of the amplification reagent. Such reagents may include lysisagents, acids, bases, surfactants, enzymes, preservatives, labels (e.g.,fluorophores), and the like. A nonexclusive set exemplary reagents areprovided in priority application PCT/US2010/02816. The reagent may bepackaged with the substrates. The reagent may also be packaged (e.g., indried form) within an area (e.g., a well) of a substrate. The kit mayalso include one or more fluids (e.g., water, buffer) that may be usedto reconstitute a reagent stored within the kit. The kit may alsoinclude a sample collection device (swab, syringe, pipette) forcollecting a sample.

As described elsewhere herein, the first population of wells comprisestwo or more wells that differ in volume from one another; similarly, thesecond population of wells comprises two or more wells that differ involume from one another. The reagent may be adapted (or selected) toparticipate in one or more of polymerase chain reaction, nucleic acidsequence based amplification, recombinase polymerase amplification,loop-mediated amplification, rolling circle amplification, helicasedependent amplification, transcription mediated amplification, multipledisplacement amplification, strand displacement amplification, or anycombination thereof.

In the disclosed kits, the at least one reagent may be disposed withinat least some of the first population of wells, the second population ofwells, or both. The reagent may be, as described elsewhere herein,present in dried form.

The first and second substrates may be planar at a region of overlapbetween the two substrates, as shown in FIGS. 2 and 6. Alternatively,the substrates may be non-planar at a region of overlap between thesubstrates. For example, the substrates may be curved, conical, or evenfrustoconical.

The disclosed kits may include a device capable of supplying or removingheat from the first and second substrates. Such devices include heaters,refrigeration devices, infrared or visible light lamps, and the like.The kits may also include a device capable of collecting an image of atleast some of the first population of wells, the second population ofwells, or both. The device may be further configured to analyze theimage and to estimate a level of an analyte present in a sample that hasbeen processed by the kit. In such embodiments, the device may beconfigured to detect the presence or absence of a target present in thefirst population of wells, the second population of wells, or both. Thedevice may be manually portable (e.g., a mobile phone, a table computer,or digital camera).

As one example, an iPhone 4S™ is useful to capture results on adisclosed device. A fluorescence readout is achieved by a standardiPhone 4S™ 8MP camera equipped with a yellow dichroic long-pass filter10CGA-530 (Newport, Franklin, Mass.). Fluorescence excitation isachieved by shining light (e.g., blue light) on a device. The light maybe applied at an oblique angle, e.g., about 30 degrees. A variety oflight sources may be used; one suitable source is a blue LED (LIU003)equipped with a blue short-pass dichroic filter FD1B (Thorlabs, Newton,N.J.). Excitation light may reach the sample by direct illumination, bymultiple reflections between the device plates, or both.

Exemplary estimation methods are described in, e.g., Shen et al., JACS2011 133: 17705-17712; Kreutz et al., Anal. Chem. 2011 83: 8158-8168;and Shen et al., Anal. Chem. 2011 83: 3533-3540, each of which isincorporated herein by reference in its entirety. These and otherestimation methods may be applied to the devices and methods presentedherein.

In another embodiment, the present disclosure provides methods ofamplifying a nucleic acid molecule. These methods suitably includecontacting (a) a sample comprising at least one nucleic acid moleculedisposed at a plurality of first areas, with (b) at least one componentof an amplification reagent disposed in a plurality of second areas; thecontacting is suitably effected by placing the first and second areasinto direct fluid communication with one another. The contacting alsosuitably includes effecting relative motion between a substratecomprising the first area with a substrate comprising the second area,and exposing the area having the at least one nucleic acid molecule toconditions effective for amplification of the at least one nucleic acidmolecule,

The amplification may be effected essentially isothermally. Variousmethods of essentially isothermal amplification are set forth elsewhereherein. The essentially isothermal amplification suitably comprisesfewer than 10 changes in temperature. As stated elsewhere herein,however, it should be understood that the disclosed devices and methodsare not limited to isothermal methods of amplification.

In some embodiments, at least two of the plurality of first areas differfrom one another in volume. In some embodiments, at least two of theplurality of second areas differ from one another in volume; in stillother embodiment, at least one first area differs in volume from atleast one second area, or any combination thereof. The differences involumes allow a user to place populations of areas (e.g., wells) intofluid communication with one another to create paired areas in registerwith one another. These paired areas (e.g., wells) then define acombined reaction region. In certain embodiments, the paired areasdefine combined reaction regions of different volumes. For example, a 1nL area may be placed pairwise into fluid communication with a 5 nL areato give rise to a combined reaction region having a volume of about 6nL. The user may also combine a 1 nL area with a 10 nL area so as togive rise to a combined reaction region having a volume of about 11 nL.In this way, a user may give rise to a set of combined reaction regionshaving different volumes (6 nL and 11 nL, in the present example).

The amplification may also be performed in a multiplexed fashion. In onesuch embodiment, amplification is performed at multiple areas. Inanother embodiment, amplification of different nucleic acids isperformed at multiple areas; a user might amplify a first nucleic acidat 10 locations, and amplify a second nucleic acid at 10 differentlocations.

The relative motion between the various areas may be linear translation,rotational motion, or nonlinear motion. The relative motion may beeffected manually (e.g., by hand). Alternatively, the relative motionmay be effected under control of a programmed (or programmable)controller. Such a controller may interface with a motor that in turneffects motion of the substrates.

In some embodiments, the first and second areas are placed into directfluid communication with one another; i.e., the areas face one another.In some embodiments, one may remove a barrier (e.g., a membrane, film,and the like) that resides between first and second areas.

A user may also estimate a level (e.g., concentration) of nucleic acidpresent in the sample. This estimation may be performed by a variety ofmethods described herein. Exemplary estimation methods are described in,e.g., Shen et al., JACS 2011 133: 17705-17712; Kreutz et al., Anal.Chem. 2011 83: 8158-8168; and Shen et al., Anal. Chem. 2011 83:3533-3540, each of which is incorporated herein by reference in itsentirety.

In some embodiments, at least 10 first areas are placed into directfluid communication with at least 10 second areas essentiallysimultaneously. In other embodiments, at least 50 first areas are placedinto direct fluid communication with at least 50 second areasessentially simultaneously. A user may place at least 500 first areasinto direct fluid communication with at least 500 second areasessentially simultaneously.

Areas that define a volume in the range of from about 0.1 nL to about1000 nL are considered particularly suitable. Volumes in the range offrom about 5 to about 10 nL are considered suitable.

In applying the disclosed methods, the area having the at least onenucleic acid molecule is estimated to contain one molecule of nucleicacid. This estimation may be performed mathematically; a user maymodulate sample concentration and area volume so as to arrive at aconfiguration wherein at least one area contains a single nucleic acidmolecule.

The disclosed methods may also include the step of amplified nucleicacid. This detection may be effected by assaying an area for thepresence of a marker (e.g., a label) that is indicative of the presenceof the nucleic acid. A user may also correlated an estimated level ofthe at least one nucleic acid in the sample to a disease state in thesource of the sample. As one example, a user may determine that thepresence of a particular nucleic acid that marks the presence of abacteria in an amplification product indicates the presence of thatbacteria in the source of the sample. A user may also isolate a nucleicacid from the sample; such isolation may include manipulating the sample(pipetting, diluting, concentrating) so as to isolate nucleic acid. Theuser may also use a capture material (e.g., silica) that adsorbs nucleicacids.

In some embodiments, the user may disposed the at least one nucleic acidat the first area. This may be effected by pipetting, spraying,injecting, and the like. A user may introduce the sample at the firstarea, introducing a amplification reagent at the second area, or both.

In some embodiments, introducing the sample at the first area comprisesexerting the sample through a conduit in fluid communication with thefirst area. The conduit may be formed in a substrate that comprises thefirst area. A user may also introduce sample at the second area; thismay be accomplished by exerting the sample through a conduit in fluidcommunication with the second area. Such a conduit may be formed in asubstrate that comprises the second area.

In some embodiments (e.g., FIG. 2), the user may distribute samplebetween the first area and a first control area. Sample at the firstcontrol area suitably remains free of contact with the at least oneamplification reagent, as shown in FIG. 2.

A user may also distribute the at least one amplification reagentbetween the second area and a second control area. The reagent (see FIG.2) in a control area may remain uncontacted with the sample.

Users may place amplification product into direct fluid communicationwith a third area. In this way, a user may create an amplificationproduct and then place that product (e.g., by effecting relative motionbetween an area where that product resides and another area thatcontains a reagent) into contact with a reagent. In this way, a user mayrealize multistep processes that create a first product and then furtherprocess that product.

The present disclosure also provides devices. The devices suitablyinclude a first substrate having a first population of areas, at leastone area of the first population of areas having at least one satellitearea disposed proximate to the at least one area, the at least onesatellite area being adapted to retain material from the at least onearea; a second substrate having a second plurality of area formedtherein, the first and second substrates being engageable with oneanother such that relative motion between the first and secondsubstrates places at least some of the first population of areas inregister with at least some of the second population of areas so as toplace the first and second areas into fluid communication with oneanother.

One such exemplary device is shown in FIG. 2. As shown in that figure, asatellite area may be adapted to retain material not retained by a firstand second area that are placed into fluid communication with oneanother. In this way, the satellite well retains excess material so asto prevent that material from interfering with operation of the device,e.g., by coming into contact with another area (e.g., well).

At least one of the first or second substrates suitably has a thicknessin the range of from about 10 micrometers to about 5000 micrometers.Thicknesses in the range of from 20 micrometers to about 100 micrometersare considered especially suitable. At least one area of the first orsecond populations includes an area that defines a volume in the rangeof from about 1 pL to about 1 microL.

The disclosed devices may be configured such that the relative motiongives rise to at least one reaction region defined by an area of thefirst population of areas in fluid communication with an area of thesecond population of areas. Such a reaction region may define a volumein the range of from about 1 pL to about 1 microL. A satellite well issuitably disposed proximate to at least one reaction region.

As described elsewhere herein and in the priority documents, the firstpopulation of areas may include two or more areas that differ from oneanother in volume. Likewise, the second population of areas may includetwo or more areas that differ from one another in volume.

The devices may be configured such that the relative motion between thesubstrates gives rise to at least about 10 reaction regions. The devicesmay be configured such that the relative motion gives rise to at leastabout 50, 100, 1000, or even about 5000 reaction regions.

The devices may also include (or be packaged with) one or more reagents.The reagents are suitably selected so as to be capable of participatingin one or more reactions that a user may effect on a sample disposed inthe disclosed devices. As one example, a device may include a reagentadapted to participate in nucleic acid amplification.

Also provided are methods. These methods effect amplification of atleast one nucleic acid target molecule, and the methods suitably includecontacting (1) a sample material disposed in a plurality of first areas,the sample material comprising a nucleic acid target, and at least oneof the first areas containing one molecule of the nucleic acid target,with (2) a reactant material disposed in a plurality of second areas,the contacting being effected by pairwise placement of at least some ofthe first areas and at least some of the second areas into direct fluidcommunication with one another, the contacting effecting amplificationof at least one nucleic acid target molecule.

In some embodiments, the sample material may include a reagent. As oneillustrative example, the sample material may include one or morenucleic acid target molecules as well as suitable amplificationreagents. The amplification may, in some cases, be essentiallyisothermal. In embodiments that include amplification, a user mayestimate the level of the at least one nucleic acid target.

In other embodiments, the reagent (disposed at the second area or areas)comprises an amplification reagent. In these embodiments, a user mayeffect amplification by using relative motion between the first andsecond areas to contact the sample with the amplification reagent. Auser may also expose an area containing the at least one nucleic acidmolecule to conditions effective for amplification of the at least onenucleic acid target so as to give rise to an amplification product. Asdescribed elsewhere herein, the amplification may be essentiallyisothermal. A user may also estimate the level of the at least onenucleic acid target.

The pairwise placement described above may be effected by relativemotion of the first and second areas. This pairwise placement may act toplace at least 10 first areas into direct fluid communication with atleast 10 second areas. It may also act to place at least 50 first areasinto direct fluid communication with at least 50 second areas. Therelative motion between the first and second areas may, as describedelsewhere herein, be effected manually by a controller, or both.

The present disclosure also provides methods. These methods includedispersing a first sample that comprises at least one molecule ofinterest among a plurality of first areas, at least one of the firstareas containing a single molecule of interest; dispersing a reactantmaterial into a plurality of second areas; and effecting pairwiseplacement of at least some of the plurality of first areas into directfluid communication with at least some of the plurality of second areasso as to contact reactant material with the first sample.

In some embodiments, the first sample is dispersed among the pluralityof the first areas at essentially the same time among the plurality offirst areas. In some embodiments, the reactant material is dispersedamong the plurality of second areas at essentially the same time amongthe plurality of second areas. The pairwise placement of the at leastsome of the plurality of first areas into direct fluid communicationwith at least some of the plurality of second areas may occur atessentially simultaneously the same time among the majority of any pairsformed by first and second areas. Some embodiments feature two or moreof foregoing.

In a particular embodiment of the disclosed methods, the first samplecomprises a reagent, and the user may effect a reaction between thereagent and at least one molecule of interest. The reaction may benucleic acid amplification, which amplification may be essentiallyisothermal. A user may also recover a product of the nucleic acidamplification; suitable recover methods are described elsewhere herein.The pairwise placement of areas into fluid communication with oneanother is suitably effected by relative motion of the first and secondareas, also as described elsewhere herein.

A user may effect a reaction between the contacted reactant material andthe first sample. Various suitable reactions are described elsewhereherein, and can include nucleic acid amplification (includingessentially isothermal amplification) of the at least one molecule ofinterest. The reactant may be an amplification reagent. A user mayrecover a product of the nucleic acid amplification, and this productmay be subjected to further processing.

The recited pairwise placement may place at least 10 first areas intodirect fluid communication with at least 10 second areas. The pairwiseplacement may place at least 50 first areas into direct fluidcommunication with at least 50 second areas.

Exemplary Embodiments Digital RPA

The following embodiments explore digital quantitative detection ofnucleic acids was achieved at the single-molecule level by chemicalinitiation of over a thousand nanoliter, sequence-specific, isothermalamplification reactions in parallel. Digital polymerase chain reaction(digital PCR), a method used for quantification of nucleic acids, countsthe presence or absence of amplification of individual molecules.Digital PCR, however, typically requires temperature cycling. This makesisothermal methods for nucleic acid amplification, such as recombinasepolymerase amplification (RPA) suitable.

A microfluidic digital RPA device is described here for simultaneousinitiation of over a thousand nanoliter RPA reactions by adding achemical initiator to each reaction compartment with a simple slippingstep after instrument-free pipette loading.

Two device designs, two-step slipping and one-step slipping, aredescribed using digital RPA. By using the disclosed devices, falsepositive results from pre-initiation of the RPA amplification reactionbefore incubation were eliminated. End-point fluorescence readout wasused for “yes or no” digital quantification. The performance of digitalRPA in the disclosed devices was shown by amplifying and counting singlemolecules of the target nucleic acid, Methicillin-resistantStaphylococcus aureus (MRSA) genomic DNA. The digital RPA on thedisclosed devices was also tolerant to fluctuations of the incubationtemperature (37-42° C.), and its performance was comparable to digitalPCR on the same device design. The digital RPA devices provide methodsto quantify nucleic acids without requiring thermal cycling or kineticmeasurements, with potential applications in diagnostics andenvironmental monitoring under resource-limited settings. The ability toinitiate thousands of chemical reactions in parallel on the nanoliterscale using solvent-resistant glass devices is useful for a broad rangeof applications.

To reduce thermal cycling, different isothermal amplification methodshave been developed, including loop-mediated amplification, nucleic acidsequence based amplification, recombinase polymerase amplification,rolling circle amplification, helicase-dependent amplification,transcription-mediated amplification, multiple displacementamplification (MDA), and strand-displacement amplification (SDA). Thesereal-time methods of isothermal amplification can be sensitive totemperature because the enzyme activity is highly temperature-dependent.To avoid effects of temperature changes and fluctuations, calibration ispreferably done in parallel to quantitatively analyze nucleic acids.Moreover, most of the methods for detection and analysis of nucleicacids using NASBA and RPA still depend on interpreting exponentialamplification profiles.

Although digital PCR typically requires thermal cycling and accuratetemperature control, the technique is straightforward because initiationof the amplification reaction is controlled by temperature. So-called“hot-start” modifications of PCR polymerases are now widely used andsubstantially eliminate any low-temperature non-specificpre-amplification. The PCR reaction mixture can be compartmentalizedprior to initiation with minimal risk of false-positives due topre-initiation. In situations where the infrastructure for thermalcycling is readily available, digital PCR is an attractive option fornucleic acid quantification. In limited-resource or point-of-caresettings, digital isothermal amplification methods that take place attemperatures near room temperature (such as RPA) are advantageousbecause they do not rely on a raised temperature for initiation, butrather rely on mixing. If the nucleic acid target is premixed with theinitiation reagent prior to compartmentalization, one might expect theamplification reaction to proceed even at room temperature and thusincrease the target count. Thus, one may compartmentalize the samplecontaining the nucleic acid target prior to adding the initiationreagents. Multistep manipulation can also be done with valves anddroplets, but such systems typically use complex control systems andinstrumentation, so it is preferably achieved on a device that does notrequire complex control systems and instrumentation.

Certain embodiments of the disclosed devices comprise two platescontaining wells and ducts that can be brought in contact and movedrelative to one another to manipulate fluids by creating and breakingfluidic paths. The pattern of wells and ducts in the two plates cancontain almost any program to manipulate fluid volumes;compartmentalizing a sample into many small volumes and mixing eachsmall volume with a reagent can be performed by simple subsequentslipping of the two plates.

Here is described devices and methods to perform digital isothermalamplification by using RPA. It is demonstrated that digital RPA does notrequire precise temperature control, as equivalent quantificationresults were obtained when quantifying MRSA gDNA at 37° C., 39° C., and42° C. One advance presented here is the capability to first confineindividual target molecules into separate reaction compartments, andthen deliver chemical initiators to initiate reaction in parallel, arequirement of digital RPA. The devices may, of course, be applied toperform other high throughput chemical reactions or screenings thatrequire multistep processes such as confinement of one reagent and thenaddition of subsequent reagents in sequence, as the digital RPAdescribed here is illustrative only.

RPA Results

The mechanism of DNA amplification and fluorescence signal generationfacilitated by RPA is described in elsewhere. RPA uses nucleoproteincomplexes consisting of oligonucleotide primers and recombinase proteinsto target binding sites within template DNA, Upon their binding, theprimers are extended by strand-displacing polymerases, thereby copyingthe target sequence. The use of primers binding to the opposing strandsof the template initiates a process of exponential DNA amplification.The generation of amplified target material can be monitored by anappropriate oligonucleotide-probe based fluorescence detection system Inthe approach used here, a fluorophore/quencher bearing probe isnucleolytically cut in response to sequence-specific binding toamplified DNA. This processing step results in a separation of thefluorophore and quencher groups, thereby leading to an increase inobservable fluorescence.

Although the RP A reaction normally proceeds at 39° C., it was firsttested to determine to what extent it would proceed at room temperature(25° C.) upon mixing of the reagents in well plates, thereforepotentially affecting the accuracy of the RPA results when performed ina digital format. The RPA solution was mixed with magnesium acetate andMethicillin-resistant Staphylococcus aureus (MRSA) genomic DNA (gDNA,final concentration of 5 pg/11 L), then immediately placed in the platereader (temperature controlled at 25° C.). The fluorescence intensityfrom wells containing gDNA template (FIG. 1, dark grey) startedincreasing within 20 min, which was different from the fluorescentintensity of the control well without magnesium acetate (FIG. 1, lightgrey) and the control well without gDNA template (FIG. 1, medium grey).

FIG. 1 shows RPA amplification of MRSA genomic DNA (5 pg/11 L) in a wellplate at 25° C. Triplicate curves (upper lines—dark grey) show that gDNAtemplate was amplified at room temperature. The control experimentwithout template (flat line—light grey) and the control experimentwithout magnesium acetate (Mg(OAc)₂, flat line) show no amplification.

This result suggested that the RPA reaction amplified the target nucleicacid template in the presence of magnesium acetate at room temperature.Therefore, to achieve digital RPA without false-positive errors, thenucleic acid template may be compartmentalized first and then magnesiumacetate is added to each individual compartment. The non-initiatingcomponents of the RP A reaction mixture (RPA enzymes, buffer, primers,and probe) can be added to the solution containing nucleic acidtemplate, to the solution of magnesium acetate, or to both.

To achieve this goal, a device was designed that featured two-stepslipping, which device was able to load and compartmentalize twodifferent reagents that could be combined by slipping (FIG. 2). Eachplate of the RPA device was designed to contain 800 wells of Type I (6nL) and 800 wells of Type II (3 nL).

Each Type II well also had two satellite wells (0.2 nL) to addresspotential thermal expansion during the temperature change from roomtemperature to 39° C. The satellite wells provided additional space forthermal expansion of the aqueous reagent within the compartment formedby overlapping the Type I and Type II wells. A total of 1,550 reactioncompartments (9 nL each) were formed by overlapping the Type I and TypeII wells contained in the facing plates (FIG. 2F, I, N). The device alsocontained 50 wells for control 1 (Type I wells. 6 nL, FIG. 2A, F, J) and50 wells for control 2 (Type H wells. 3 nL, FIG. 2A, F.

The digital RPA device was assembled by combining the top plate (FIG.2A) and bottom plate (FIG. 2B) with a thin layer of tetradecane betweenas the lubricating fluid. The lubricating fluid preventedcross-contamination and evaporation of the aqueous sample duringincubation. The first continuous fluidic path was formed by overlappingthe Type I wells in the two plates (FIG. 2C). RPA Reaction Mixture 1,containing RPA primers and probe, MRSA gDNA, and re-hydrated RPA enzymemixture, but no magnesium acetate, was loaded by pipetting (FIG. 2D, K).This RPA device was designed to be filled via dead-end filling,therefore, the speed of sample injection need not necessarily becontrolled so long as the applied pressure is lower than the leakingpressure.

The two plates were then slipped relative to one another tocompartmentalize RPA Reaction Mixture 1, simultaneously stochasticallyconfining the gDNA template in the Type I wells and forming the secondfluidic path by overlapping the Type II wells (FIG. 2E, L). RPA ReactionMixture 2, which contained no gDNA and contained magnesium acetate atthree fold higher concentration than required for the bulk reaction (3×,so the final concentration of magnesium acetate after mixing would beIX), RP A primers and probe, and re-hydrated RPA enzyme, was also loadedinto the chip by pipetting (FIG. 2E, M). Finally, the two plates wereslipped relative to one another to overlap the Type I wells with theType II wells in the facing plates, delivering the magnesium acetate inReaction Mixture 2 to all 1550 of the Type I wells simultaneously andinitiating the reaction FIG. 2F, N; FIG. 9). The digital RPA device wasthen placed on a flat metal adapter and incubated at 39° C. for 1 hour.Type I wells for Control 1 contained only Reaction Mixture 1 (negativecontrol, no magnesium acetate), and Type II wells for Control 2contained only Reaction Mixture 2 (negative control, no nucleic acidtemplate).

FIGS. 2A-N illustrate a schematic drawing of the two-step device fordigital RPA. FIG. 2A) shows a top plate of the device. A zoomed inschematic drawing shows the geometry of Type I, Type II and satellitewells. 2B) Bottom plate of the device. 2C) Assembly of top and bottomplates to establish the first continuous fluidic path of Type I wells.2D) Loading of the first reagent, Reaction Mixture 1 (dark grey). 2E)Slipping breaks the first fluidic path and compartmentalizes the loadedreagent. At the same time, the second fluidic path is formed byconnecting Type II wells. The second reagent, Reaction Mixture 2 (lightgrey), is loaded through a second inlet. 2F) A second slipping stepcompartmentalizes Reaction Mixture 2 into the Type II wells and overlapsthe Type II wells with the Type I wells. The two reagents are mixedwithin the reaction compartments. 2G) Microphotograph shows the entiredigital RPA device next to a United States quarter coin for scale. 2H,2I, 2J) Food dyes were loaded into the device to demonstrate loading andmixing. 2H) Zoomed in view of Type II wells for Control 2 (no template),loaded with blue food dye. 2I) Zoomed in view of reaction wells(overlapping Type I and Type II wells) containing mixed blue and orangefood dye. 2H) Zoomed in view of Type I wells for Control 1 (no magnesiumacetate), loaded with orange food dye. (2K, 2L, 2M, 2N) Experiments withfood dye demonstrate the procedures described in panels 2D, 2E, 2F ofthe figure.

A digital RPA device was applied to a sample containing a 1:104 dilutionof 5 ng/11 L of stock MRSA gDNA. The stock gDNA was purified from MRSAculture, and the optical density of the purified nucleic acid productwas measured spectrophotometrically. At this concentration, the averagecopy number of gDNA per well was expected to be less than 1, andsingle-copy RPA was achieved. The reaction solution of RPA was made fromrehydrating the lyophilized reagent, and was heterogeneous:microparticles of various sizes and shapes were still present even aftersonication and vortexing the solution (FIG. 3A). A linescan of thefluorescence intensity of wells from the digital RPA device before andafter incubation at 39° C. (FIG. 3) shows that the fluorescenceintensity of a positive well increased significantly compared to anegative well (FIGS. 3A-B) and the control wells (FIGS. 3C-F) afterincubation for one hour. The number and the size of microparticlesdecreased after incubation, which may be due to further dissolution ofthe microparticles during incubation at 39° C. There was no significantincrease of fluorescence intensity from control wells without magnesiumacetate (representative Control well 1, FIGS. 3C-D) and without gDNAtemplate (representative Control well 2, FIGS. 3E-F). Only the endpointfluorescent intensity was monitored in this experiment. Theamplification signal may be observed in less than 30 min. A real-timefluorescence detector can be used to further investigate the uniformityof amplification and to optimize the total time required for incubation.

FIGS. 3A-F illustrate fluorescence microphotographs and linescans of RPAon the device before and after incubation at 39° C. (3A-3B) Negative(left) and positive (right) sample wells: (3A) before incubation, thefluorescence intensity in both wells is the same. (3B) After incubation,the integrated fluorescence intensity in the positive well (right) issignificantly higher compared to the negative well (left). (3C-3D)Control well 1, containing no magnesium acetate, before (3C) and after(3D) incubation shows no significant increase in fluorescence intensity.3E-3F) Control well 2, containing gDNA template, before (3E) and after(3F) incubation also shows no significant increase in fluorescenceintensity.

Performance of the digital RPA device was explored using a serialdilution of the MRSA gDNA stock solution at five orders of magnitude,from 1:10 dilution to 1:10⁵ dilution. FIGS. 4A-4E illustrate thefraction of positive wells on the RPA device at a variety of dilutions.For example, FIG. 4A provides a dilution of 1:10; FIG. 4B provides adilution of 1:10²; FIG. 4C provides a dilution of 1:10³; FIG. 4Dprovides a dilution of 1:10⁴; and FIG. 4E provides a dilution of 1:10⁵.As the gDNA template was diluted, the fraction of positive wells on theRPA device decreased proportionally after incubation (FIGS. 4A-4E andFIG. 5). No evidence of contamination was observed as no false positiveswere observed in the control (no DNA template, FIG. 4F). The experimentswere repeated three times at each concentration of gDNA to demonstratethe robustness and reproducibility of the digital RPA on the device(FIG. 5). The data from RPA on the device with serial diluted gDNAtemplate followed a. Poisson distribution. A statistical analysis of theresults from digital nucleic acid amplification on the device wasperformed as previously described (Lab Chip 2010, 10, 2666-2672). Byfitting the results from the 1:10³, 1:10⁴, and 1:10⁵ dilutions to aPoisson distribution (FIG. 5), the concentration of stock MRSA gDNA wascharacterized to be approximately 10 million copies/mL. A 95% confidenceinterval for the fitted Poisson distribution was calculated based onmethods previously presented (Lab Chip 2010, 10, 2666-2672) (FIG. 5,dashed lines).

FIG. 4 shows a digital RPA on a device with different concentration ofMRSA gDNA. A-E) Digital RPA on the device with a serial dilution oftarget DNA template ranging from 1:10 to 1:10⁵ of a 5 ng/11 L stocksolution. (F) Control, no wells showed positive signal when no targetDNA was loaded.

FIG. 5 illustrates quantified results of digital RP A on the device.Experimental average of the number of positive wells was plotted as afunction of the dilution of the MRSA gDNA sample. Error bars representstandard deviation of the experiment (n=3). The black solid linerepresents the Poisson distribution obtained by fitting the data fromthe 1:10⁵, 1:10⁴, and 1:10³ dilution of template. Gray dash linesrepresent the 95% confidence interval for the fitted Poissondistribution.

The device design described above uses a two-step procedure for loadingreagents: the two reagents can be loaded independently of one another,an attractive capability for general parallel processing of samples andreactions. Incubation or thermal cycling can be performed afterconfining the target molecules or the first reagent into individualreaction compartments, then additional reagents can be delivered (e.g.,reagents for readout) into each compartment in parallel. This featurealso facilitates quality control during development of new methods.Digital RPA typically requires this parallel processing of reactions,but does not typically specifically require two-step processing. Alsopresented here is a simplified device that does not independentlycontrol reagents but instead allows compartmentalization and mixing ofthe two reaction mixtures in parallel by one-step slipping aftersimultaneous introduction of the reagents (FIGS. 6A-6E). Digital RPAwith a 1:10⁴ dilution of MRSA gDNA template is demonstrated on thisone-step device, and the result is consistent with the two-step devices(FIG. 6 F, compare to FIG. 7B, n˜3, p>0.2)

FIGS. 6A-6F illustrate a device for one-step digital RPA 6A-6C)Schematic drawings of the device: 6A) Assembly of top and bottom platesto establish the continuous fluidic path for both Type I wells and TypeII wells. 6B) The first solution, Reaction Mixture 1 (dark grey), andsecond solution, Reaction Mixture 2 (light grey), were introducedsimultaneously into the device. 6C) Slipping breaks both fluidic pathsand compartmentalizes the loaded reagent. At the same time, the Type Iwells were overlaid with Type II wells to initiate the reaction. 6D, 6E)Microphotographs showing food dyes loaded into the device to demonstrateloading and mixing. 6F) Zoomed-in fluorescent image of a fraction ofdigital RPA on one-step device with a 1:104 dilution of MRSA gDNAtemplate after incubation at 39° C.

As shown, RPA can be initiated at room temperature (˜25° C.) aftermagnesium acetate is added (FIG. 1). To achieve digital RPA, thereaction mixture containing target nucleic acid template may bepreferably separated into isolated reaction compartments beforemagnesium acetate IS added. This was demonstrated quantitatively on thedisclosed devices. Instead of mixing Reaction Mixture 1 (withoutmagnesium acetate) with Reaction Mixture 2 (with magnesium acetate)on-chip, the reaction solution (containing a 1:104 dilution of gDNA) wasmixed with magnesium acetate to initiate the reaction off-chip, andincubated the solution at room temperature (˜25° C.) for 1 minute. Onemay refer to this off-chip mixing and incubation as the “pre-initiated”reaction solution. The pre-initiated reaction solution was then injectedinto the two-step digital RPA device at room temperature through theType I wells, and slipped to compartmentalize. The injection step tookaround 4 minutes. A second solution that contained magnesium acetate,RPA primers and probe, and re-hydrated RPA enzyme was loaded into theType II wells as described above.

Following that, the Type I and Type II wells were overlaid by slippingthe top plate relative to the bottom plate. The device was thenincubated at 39° C. for 1 hour. These results were compared to resultsobtained without pre-initiating the solution with magnesium acetateoff-chip (from experiments shown in FIGS. 4 and 5). The fraction ofpositive wells from the pre-initiated sample was significantly higherthan in the sample without pre-initiation (FIG. 7A, n=3, p<0.01).Without being bound to any single theory, one may attribute the largestandard deviation in the measurement of the pre-initiated sample to thevariation in loading time changing the extent of reaction prior tocompartmentalization; reaction taking place during loading is alsoconsistent with the “streaky” distribution of the positive wells inthese experiments (see FIG. 10). These results demonstrate thatcompartmentalization followed by chemical initiation of the RP Areaction is preferred for obtaining quantitative results using digitalRPA.

FIGS. 7A and 7B illustrate: 7A) Comparing on-chip mixing (nopre-initiation) to pre-initiation with magnesium acetate on the two-stepdigital RPA device. The sample with pre-initiation with magnesiumacetate prior to compartmentalization shows a higher fraction ofpositive wells, indicating that compartmentalization prior to theaddition of magnesium acetate is preferred for achieving accuratedigital RPA; 7B) Comparing two-step digital RPA, one-step digital RP Aand digital PCR Samples containing MRSA gDNA at the same dilution(1:10⁴) were quantified using two-step digital RP A (as in FIG. 4)(left, n=3), one-step digital RPA (as in FIG. 6) (middle, n=5), anddigital PCR (right, n=3) on the RPA device. Error bars representstandard deviation.

To further demonstrate the performance of digital RPA on the devices, wecompared experiments of digital RP A to experiments of digital PCR usingthe same concentration of MRSA gDNA on the same device (1:10⁴ dilution,see also FIG. 11). The same mecA gene in MRSA gDNA was targeted forquantification in both methods. The average results from two-stepdigital RPA and one-step digital RPA were not significantly different(p>0.2, n≧3) than from digital PCR (FIG. 7B). Because RPA does notbenefit from the high temperature step employed in PCR, one potentialconcern regarding the use of digital RP A is sensitivity to secondarystructures of nucleic acids or to contamination with nucleic-acidbinding proteins; this could lead to lower “counts” of nucleic acids. Toaddress this concern, RPA was designed to operate in the presence ofcomparatively large amounts of gp32, the single-strand binding proteinfrom T4-like bacteriophages. Gp32 has been reported to bind ssDNA and“melt” secondary DNA structures. Gp32 It has also been used as a commonenhancer of various molecular biology techniques, including PCR andreverse transcription.⁵⁴

FIGS. 8A-8D show RPA two-step devices for amplification of MRSA gDNAwith incubation at different temperatures. FIGS. 8A-8C) Representativefluorescent images of RP A for MRSA gDNA with dilution of 1:10⁴ at 3rC(8A), 39° C. (8B), and 42° C. (8C). 8D) Histogram showing number ofpositive wells from RPA on the devices at different incubationtemperatures. Error bars represent standard deviation of the experiment(p>0.2, n≧4).

The digital RPA device typically depends on the endpoint fluorescencereading of either “0” or “1”, unlike real-time PCR and real-time RPAthat monitor the change of fluorescence intensity over time. Because theenzyme activity depends on the working temperature, the temperature candramatically affect the amplification speed in real-time RPA Therefore,real-time amplification methods require accurate control of temperatureand careful calibration for quantitative analysis. This may makereal-time RPA less applicable in point-of-care diagnostics in resourcelimited settings. Because the digital devices detects the endpointreadout instead of real-time changes of fluorescent intensity, thedigital RPA device may be more tolerant to temperature fluctuations thanreal-time methods. Amplifications of MRSA gDNA at 37° C., 39° C., and42° C. were not significantly different (FIG. 8, p>0.2, n≧4). Increasingthe temperature decreased the required incubation time, and quantitativeresults were, for example, achieved in as short as 30 min withincubation under 42° C.

Results Summary

Parallel initiation of pre-compartmentalized reactions on the discloseddevices is suitable for isothermal nucleic acid quantification by usingrecombinase polymerase amplification (RPA) at 39° C. in a digitalformat. The RPA reaction will start even at room temperature once themagnesium acetate is added into the reaction mixture, increasing thenumber of false positives in digital RPA if the reaction mixture iscompartmentalized after off-chip mixing of all reagents with the nucleicacid template. The digital devices addressed this issue by separatingthe reaction mixture containing nucleic acid template into individualcompartments, in the absence of magnesium acetate, and then deliveringmagnesium acetate to all compartments simultaneously by slipping. Aone-step device was also demonstrated using digital RPA, and the resultwas consistent with the results obtained on the two-step device. Thedigital RPA device was also demonstrated to be robust in the presence ofsmall perturbations of incubation temperature from 37-42° C. The digitalRPA device was designed to contain 1550 reaction compartments of 9 nLeach, with two additional sets of wells for controls (50 wells for eachcontrol), giving a potential for, in certain embodiments, a detectionlimit of 300 copies/mL and dynamic range of 1400 to 1,000,000 copies/mLwith three-fold resolution, calculated using the method describedpreviously. The RPA reaction was robust and free of cross-contaminationon the device. However, microparticles were present in the reactionmixture even after vortexing and sonication. A real-time imaging systemcan also be used. No false positive results were observed in theexperiments. Incorporation of a reverse-transcription step with RPA canexpand the applicability of the digital RPA devices for quantitativeanalysis of viral loads in resource-limited areas. The disclosed methodsprovide a platform for quantification of nucleic acids under resourcelimited settings and in the clinic, where digital PCR and real time PCRmay not be available due to limited infrastructure; in the disclosedmethods, reagents and template can be loaded as one solution and onlyone slip is required.

Chemicals and Materials—RPA

All salts and solvents purchased from commercial sources were used asreceived unless otherwise stated. The TwistAmp exo kit for RP A waspurchased from TwistDx Limited (Cambridge, United Kingdom). The RP Aprimers and probe for detection of MRSA mecA gene were generouslyprovided by TwistDx Limited. Bovine serum albumin (BSA) solution wasordered from Roche Diagnostics (Indianapolis, Ind.). Tetradecane,chloroform, acetone, ethanol, and DEPC-treated and nuclease free waterwere obtained from Fisher Scientific (Hanover Park, Ill.).Dichlorodimethylsilane was purchased from Sigma-Aldrich (St. Louis,Mo.). Soda-lime glass plates coated with photoresist and chromium wereordered from Telic Company (Valencia, Calif.). Spectrum food colors(brown and blue food dye) were obtained from August Thomsen Corp (GlenCove, N.Y.). Photomasks were ordered from CADI Art Services, Inc.(Bandon, Oreg.). PCR tubes and barrier pipette tips were purchased fromMolecular BioProducts (San Diego, Calif.). All PCR primers werepurchased from Integrated DNA Technologies (Coralville, Iowa). SsoFastEvaGreen Supermix (2×) was obtained from Bio-Rad Laboratories (Hercules,Calif.). Methicillin-resistant Staphylococcus aureus [MRSA], ATCC 43300,was purchased from American Type Culture Collection (Manassas, Va.) andMRSA gDNA was purified according to the manufacturer's recommendationsusing Qiagene Puregene Yeast/Bact. Kit A obtained from Qiagen (Valencia,Calif.).

Fabrication of Devices for Digital RPA

The procedure for fabrication oft from soda lime glass was based onmethods developed previously (Lab Chip 2009, 9, 2286-2292). To fabricatedevices with wells of different depths, the following procedures wereused: 1) The glass plate coated with chromium and photoresist wasaligned with a photomask containing the design for Type I wells, Type IIwells, and ducts by using a mask aligner, then the photoresist layer wasexposed to UV light using standard exposure protocols. 2) Afterexposure, the glass plate was immersed in 0.1 mol/L NaOH solution toimmediately remove the photoresist from exposed areas. 3) The exposedunderlying chromium layer was removed by using a chromium etchant (asolution of 0.6:0.365 mol/L HC10₄I(NH₄)₂Ce(NO₃)₆). 4) The glass platewas then thoroughly rinsed with Millipore water and dried with nitrogengas. 5) The glass plate was then immersed in a glass etching solution(1:0.5:0.75 mol/L HF/NH₄F/HNO₃) to etch the glass surface wherephotoresist layer and chromium coating were removed in the previoussteps. 6) The glass plate was thoroughly washed with Millipore water anddried with nitrogen gas. 7) The glass plate was aligned with a secondphotomask containing the design of satellite wells and was exposed to UVlight by using the standard exposure protocols. Then steps 2) to 6) wererepeated. Finally, the remaining photoresist was removed by usingacetone, and the underlying chromium layer was removed by using thechromium etchant. The etched depth was controlled by the etching timeand speed, which was controlled by the etching temperature. The Type Iand Type II wells were etched to be 50 μm deep, and the satellite wellswere etched to be 15 μm deep. The volume of Type I wells, Type II wells,and satellite wells were 6 nL, 3 nL, and 0.2 nL respectively.

The glass plate was oxidized in a plasma cleaner (Structure Probe, Inc.,West Chester, Pa.) for 10 minutes and then immediately transferred intoa desiccator (Fisher Scientific, Hanover Park, Ill.).Dichlorodimethylsilane (50 11 L) was injected into the desiccator andthen a vacuum was applied to perform gas-phase silanization for onehour. The silanized glass plate was thoroughly cleaned with chloroform,acetone, and ethanol, and then dried with nitrogen gas. The silanizedglass plate was used for digital RP A experiments within one day. Theglass plate was reused after thoroughly cleaning with piranha solution(3:1 sulfuric acid:hydrogen peroxide) and silanized with the proceduredescribed above.

Assembling the Devices

The devices were assembled under tetradecane. The tetradecane wasde-gassed before digital RPA experiments. The bottom plate was firstimmersed into tetradecane in a Petri dish, with the patterned wellsfacing up. The top plate was then immersed into tetradecane and placedon top of the bottom plate with the patterned side facing down. The twoplates were aligned under a stereoscope (Leica, Germany) as shown inFIG. 1 and stabilized using binder clips.

Digital RPA on Two-Step Devices with on-Device Initiation

RPA master mixture was prepared by rehydrating the lyophilized enzymemixture in 29.5 μL of rehydration buffer and 10 μL of water, then adding3.5 μL each of RPA primers A and B (10 μM each) and 1 μL of themecA-specific probe (TwistDx Ltd). The solution was pulse-vortexed threetimes and sonicated in a FS60H (Fisher Scientific) at room temperaturefor 10 minutes. 5 μl of BSA solution (20 mg/ml) was added to the RPAmaster mixture. For experiments with on-chip initiation of digital RPA(FIGS. 3, 4, 5, 6, 7), 1.5 μL of MRSA gDNA template solution (1:10⁵ to1:10 dilution) was added to 28.5 μL of the RPA master mixture asReaction Mixture 1 (FIG. 1, medium grey line). 4 μL of 280 mM ofMg(OAc)₂ solution was added to 15 μL of the RPA master mixture asReaction Mixture 2 (FIG. 1, light grey line). Reaction Mixture 1 wasloaded into the device by pipetting (FIGS. 2C-D), and then the top platewas slipped relative to the bottom plate to compartmentalize the gDNAtemplate in Reaction Mixture 1. Then Reaction Mixture 2 was injectedinto the device (FIG. 2E), and the top plate was slipped again in thesame direction relative to the bottom plate to overlay the Type I andType II wells and to initiate the digital RPA reaction simultaneously(FIG. 2F).

FIGS. 9A-9E illustrate a food dye experiment demonstrated the operationof slipping for digital RPA device. 9A) The top and bottom plates of thedigital RPA devices were aligned to form the continuous fluidic path byoverlapping the Type I wells. 9B) The first reagent (light grey) wasloaded into the device by pipetting. 9C) The top plate was slippedrelative to the bottom plate to compartmentalize the reagent loaded inthe Type I wells, and a second fluidic path was formed by overlappingthe Type 11 wells 9D) The second reagent (dark grey) was loaded into thedevice by pipetting. 9E) The top plate was slipped again relative to thebottom plate, and the Type I and Type II wells were overlaid to combinethe two reagents. FIGS. 9A-9E show the step-by-step loading procedureusing food dyes. The device was placed on a metal adaptor and incubatedfor 1 hour at 39° C. (experiments in FIG. 3, 4, 5, 6, 8B, 8D).

RPA on a One-Step Device with on-Device Initiation

RPA master mixture was prepared by rehydrating the lyophilized enzymemixture in 29.5 μL of rehydration buffer and 10 μL of water, then adding3.5 μL each of RPA primers A and B (10 μM each) and 1 μL of themecA-specific probe (TwistDx Ltd). The solution was pulse-vortexed threetimes and sonicated in a FS60H (Fisher Scientific) at room temperaturefor 10 minutes. 5 μL of BSA solution (20 mg/ml) was added to the RP Amaster mixture. 1.5 μL of MRSA gDNA template solution (1:10⁴ dilution)was added to 28.5 μL of the RPA master mixture as Reaction Mixture 1(FIG. 1, light grey line). 4 μL of 280 mM of Mg(OAc)₂ solution was addedto 15 11 L of the RPA master mixture as Reaction Mixture 2 (FIG. 1,medium grey line). Reaction Mixture 1 and Reaction Mixture 2 wereintroduced simultaneously into the one-step device by applying pressureas described before.² One slipping step broke both fluidic paths andcompartmentalized the loaded reagent. At the same time, the Type I wellswere overlaid with Type II wells to initiate the reaction (FIG. 6).

RPA on the Two-Step Device with on-Device—Initiation

For experiments with pre-initiation (FIGS. 1, 7 A, and 10), 5 μL of BSAsolution (20 mg/mL) and 4 μL of Mg(OAc)₂ solution (280 mM) were added to48 μL of the RPA master mixture. A solution of 1.5 μL of MRSA gDNAtemplate solution (1:10⁴ dilution) was added to 28.5 μL of this reagentmixture as Reaction Mixture 1. The remaining solution was treated asReaction Mixture 2. Reaction Mixture 1 was incubated at room temperature(approximately 25° C.) for 1 min, and then injected into the device bypipetting (FIG. 2C-D). The entire loading procedure took 4 minutes underroom temperature. The top plate was slipped relative to the bottom plateto compartmentalize the RPA solution (FIG. 2E). Reaction Mixture 2 wasthen loaded into the device by pipetting (FIG. 2E). The top plate wasslipped again relative to the bottom plate and the Type I wells wereoverlaid with Type II wells (FIG. 2F) The device was then placed on anadaptor for incubation at 39° C. for one hour.

FIG. 10 shows a “streaky” distribution of positive wells was obtainedwhen RPA is pre-initiated off-chip for one minute and loaded onto thechip via pipetting over 4 minutes. This result indicates that theamplification reaction is proceeding as the reaction mixture is beingloaded. MRSA gDNA (at concentration of 1:104 dilution) was pre-mixedwith Reaction Mixture 1 (containing magnesium acetate). Thepre-initiated mixture was loaded into Type I wells from the right sideof the digital RP A device as described in FIG. 2.

Quantitative RPA by Using a Plate Reader

RPA master mixture was prepared as described above. 5 μL of BSA solution(20 mg/mL) and 4 μL of Mg(OAc)₂ solution (280 mM) were added to 48 μL ofthe RPA master mixture. Then 19 μL of the reaction mixture was placed ina well of a 96 well plate, and 1 μL of MRSA gDNA template solution(1:10³ dilution) was added to each well. The well plate was immediatelyplaced in a plate reader (BMG LABTECH, Germany) with temperaturecontrolled at 25° C. For the control experiment without template, afterloading the reaction mixture into a 96 well plate, 1 μL of water wasadded to each well instead of gDNA template solution (FIG. 1, mediumgrey line). For the control experiment without Mg(OAc)₂ solution, 54 ofBSA solution (20 mg/mL) and 4 μL of water were added to the RPA mastermixture. Then 19 μL of the reaction mixture was placed in a well of a 96well plate, and 1 μL of MRSA gDNA template solution (1:10³ dilution) wasadded to each well (FIG. 1, light grey line). Fluorescence intensity wasacquired every minute for two hours. A shaking step of 2 seconds wasapplied after each acquisition cycle.

Digital PCR on the Disclosed Devices

A digital RPA device was designed to be compatible to perform digitalPCR as well (FIGS. 11A-11E). For digital PCR, the PCR reaction mastermixture consisted of 20 μL of SsoFast EvaGreen SuperMix (2×), 2 μL ofBSA solution (20 mg/mL), 15 μL of water, and 1 μL of each forward andreverse primers (10 μM each). A solution of 1.5 μL of MRSA gDNA template(1:10⁴ dilution) was added to 28.5 μL of the above reaction mixture. Theprimers for detection of mecA gene in MRSA gDNA were: primer 1, CAA GATATG AAG TGG TAA ATG GT; primer 2, TTT ACG ACT TGT TGC ATACCATC.

FIGS. 11A-11E provide schematic drawing showing the procedures toperform digital PCR by using the two-step device. 11A) top and 11B)bottom plates of the device. 11C) Assembly of top and bottom plates toestablish the first continuous fluidic path of Type I wells. 11D)Loading of the PCR reagent (red). 11E) One-step slipping tocompartmentalize the PCR reagent and overlap with Type II wells.

The PCR reaction mixture was injected into the device to fill all theType I wells (FIG. 11D). Without loading the second reagent, the topplate was slipped relative to the bottom plate to directly overlay theType I wells with the Type II wells (FIG. 11E). The Type II wells, whichwere previously filled with tetradecane during assembly of the device,offered additional volume for thermal expansion during PCR thermalcycling. The device was then placed on an adaptor in the Mastercyclerfor thermal cycling.

An initial step at 95° C. (2 min) was used to activate the enzyme forreaction. Next, a total of 35 cycles of amplification were performed:denaturation step of 1 min at 95° C., annealing step of 30 sec at 55°C., and a DNA synthesis step of 30 sec at 72° C. After the final cycle,a final DNA extension step was performed for 5 min at 72° C.

Image Acquisition and Analysis

All fluorescence images were acquired by using a Leica DMI 6000 Bepi-fluorescence microscope (Leica Microsystems, Germany) with a 5× I0.15 NA objective and L5 filter. All fluorescence images were correctedby a background image obtained with a standard fluorescent slide.Fluorescence images were stitched together by using MetaMorph software(Molecular Devices, Sunnyvale, Calif.).

NASBA Amplification—Signal Generation

Nucleic acid sequence based amplification (NASBA) is an isothermalamplification method that functions at around 41° C. and can reach veryhigh levels of amplification (>10¹⁰). The mechanism is described in FIG.13. The RNA product makes this method particularly appealing as itshould facilitate hybridization-based detection methods.

In one embodiment, digital NASBA is performed by sequential reactions onmultiple compartments. The first compartments are created with NASBAreagent (listed in Table 1) with exception of enzyme and molecularbeacon after denaturation at 65 degrees Celsius. Enzyme is added to thefirst compartments to initiate NASBA reaction. This first reaction canbe carried out by incubation at 41 degrees Celsius to amplify moleculeof interest. Second compartments of molecular beacon solution arecreated and combined with the first compartments to carry out the secondreaction. The second reaction is a detection reaction of amplificationproduct.

In another embodiment, multiplex NASBA can be carried out by sequentialreactions on multiple compartments. primers can be patterned in reactionareas (wells or surfaces) of one of the disclosed devices, for example,as described in Analytical Chemistry 2010 82:4606-4612, or the primersolution can be user-loaded as described in JACS 2010 132: 106-111. TheNASBA reagent containing molecule of interest is compartmentalized andmixed with preloaded primers and the first reaction of amplification iscarried out. The second compartments containing detection reagent, suchas different molecule beacons, are created and combined with the firstcompartments to carry out the second reaction of detection.

Digital NASBA is now described here in further detail. Although thedigitization process was performed in the disclosed devices, butfunctions in other platforms as well. See FIG. 14 for a schematic of thetwo stage device used herein. Fluorescent beacons were used for readout.HIV RNA was used for proof-of-principle work and previously publishedprimers and probes were used as a starting point. (de Baar, M. P., etal. One-tube real-time isothermal amplification assay to identify anddistinguish human immunodeficiency virus type 1 subtypes A, B, and C andcirculating recombinant forms AE and AG. J. Clin. Microbiol. 39,1895-1902 (2001).) The originally published beacon V2 (after correctionfor a mutation in the used template) had its hairpin modified to improvehybridization resulting in V3 which showed a signal:background ratio of20-40 depending on detection method used (Table 1, shown in FIG. 15) (40fold using nanodrop, closer to 20 fold on chip or using plate reader).

FIG. 16 provides an example of the digital readout in the discloseddevices, with a linescan showing the increased signal from positivewells. There is good agreement between results from digital NASBA anddigital RT-PCR (FIG. 17). In these experiments the NASBA reactions wereinitiated on-chip, where the solution containing beacon and enzyme wasintroduced to the solution containing template and primers after thesolutions were first isolated on chip. This was to prevent anybackground amplification from altering the expected concentration. Thispreisolated amplification was characterized by storing premixedsolutions at different temperatures (FIG. 18). On-chip initiation may berequired in limited resource settings where temperature control ofsamples may not be stable, but if suitable temperature control existsthen use of a premixed sample may simplify device design.

Visual Readout—NASBA

Several visual readout methods based on hybridization of the RNA NASBAproduct are suitable. Simple concentration of functionalized goldnanoparticles (AuNP's) such as in many lateral flow assays is oneapproach. Alternatively, nanoparticle-based aggregation, where twodifferent types of AuNP's that would recognize different sequences onthe product RNA, may be used to observe a color change to detectpresence of product. The small scale of the wells may make theseapproaches impractical from an intensity standpoint, so additionalamplification methods are also being explored.

One approach is silver amplification where AuNP's catalyze thedeposition of Ag(s) from solution. The deposition chemistry wasoptimized to maximize signal in the presence of AuNP and minimizebackground; in this digitized approach, the presence of product is a keyoutput. Through screening of various thiols, it was determined that˜3-mercaptoethanol (˜3-ME) can suppress AuNP catalyzed amplification atlow AuNP concentrations, and also further suppresses the backgroundreaction (when no nanoparticles were present). A methoxy-terminated PEG5000 thiol (PEGThiol) had no effect on the nanoparticle free reaction,but when nanoparticles were present it resulted in very strong signalgeneration (FIG. 19b ), helping to generate more uniform intensityregardless of AuNP concentration. In well-plates the use of 13-ME andPEG-thiol could easily differentiate 25 pM from the background, and maygive complete signal generation in under 10 minutes for concentrationsas low as 100 pM. Even after one hour, the background generated littlesignal (FIG. 19a ). The rate of the reaction could also be altered bythe Ag:H2Q ratio and concentration. A 1:4 ratio seemed to give strongestsignal and at concentrations of 5 and 20 mM respectively the stablebackground reaction was achieved. Experiments using an antibody-basedhybridization system showed that the desired properties were maintainedeven upon conjugation of the AuNP to magnetic nanoparticles, and thatsignal was generated only from high target concentrations (FIG. 19b ).Experiments show that silver amplification can occur on the discloseddevices and be easily distinguished from negative controls (FIG. 19c ).

NASBA Amplification—Additional Disclosure

Digital Nucleic Acid Sequence-Based Amplification (NASBA) can beperformed at the single-molecule level on the disclosed devices. In oneembodiment, the reagents for performing digital NASBA can be mixedtogether on a device as disclosed herein. For example, the amounts andtypes of reagents listed in Table 1 below have been used to performdigital NASBA on a disclosed device:

2x 5 μM Buffer + NASBA primer 2% (20 mg/mL) 0.1% 8x 4 μM Buffer mix BSABuffer Split BSA Template Enzyme Beacon Control 5 0.8 1 1.95 rest 1.25 —— — Template 20 3.2 4 7.8 35 — 5 — — Combined 25 4 5 9.75 Enzyme 25 — 52.5 — — 12.5 5

In another embodiment, the reagents listed in Table 1, with theexception of enzyme, can be pre-mixed and heated to 65 degrees Celsiusbefore adding the mixture to the device, where it is combined withenzyme.

In another embodiment, the reagents listed in Table 1, with theexception of the molecular beacon, can be pre-mixed and heated to 65degrees Celsius before adding the mixture to the device, where it iscombined with the molecular beacon.

In yet another embodiment, the reagents for performing digital NASBA canbe pre-mixed before adding the mixture to the device. For example, theamounts and types of reagents listed in Table 1 have been used toperform digital NASBA on the disclosed devices:

In experiments performed using the reagents in Table 1, the NASBA buffertogether with Accusphere, both of which were obtained from LifeSciencesAdvanced Technologies Inc., were vortexed and then heated to 41° C. for5-10 minutes then vortexed again prior to addition to tubes to maximizesolubility and solution uniformity. The other buffer used was 10 mM TrispH 8.5, 75 mM NaCl, 50 mcM EDTA. It was made using DNA grade H₂O(BP2470-1 from Fisher), and was filtered through a 0.45 μm filter andautoclaved. All DNA primers, probes, and model templates were orderedfrom IDTDNA.com. They were dissolved to 100 μm in DNA grade H₂O.Dilutions down to about 5 μM were made in Buffer. Dilutions below 1 μMwere made in Buffer+Bovine Serum Albumin (BSA, 0.1%). The final primerand molecular beacon concentrations were 200 nM. 90 minutes of reactiontime at 41 degrees Celsius was sufficient for reactions on the discloseddevices.

For performing digital NASBA on HIV viral RNA, the primers that wereused have the following sequences:

5′ TAATACGACTCACTATAGGGTGCTATGTCACTTCCCCTTGGTTCTCT CA 5′GTGGTGGGATATCAAGCAGCCATGCAAA

The molecular beacon that was used had the following sequence:

5′-/56-FAM/CGGATGCTGCAGAATGGGATACAGTGCATCC/3IABkFQ/ 3′

This beacon was found in experiments to produce a 20 fold increase offluorescence intensity on chip or using a plate reader and a 40 foldincrease of fluorescence intensity on nanodrop. Another beacon with thesame sequence, but replacing FAM with Cy5 and replacing 3IABkFQ with3BHQ_2, has also been used. 90 minutes appears to be sufficient reactiontime for on-chip experiments though shorter times may be possible (<1hr) based on real time well plate experiments.

FIG. 12 shows experimental results showing digital reverse-transcriptionpolymerase chain reaction (RT-PCR) and digital NASBA performed on thedisclosed devices using the same template and initial concentration,showing parallel results at three different concentrations. The devicesused herein were comparable to those published devices described inAnalytical Chemistry 2011 83:3533-3540. The device used for NASBAapplications included two sets of wells and enabled performing thereagent mixing step on the disclosed devices. Instead of having 6 nLtype I and 3 nL type II wells, as in the published devices, the deviceused for these NASBA experiments had two types of wells of the same size(2.6 nL). The device's plates could be slipped relative to one anotherto enable functions in four different positions: position 1 for loadingsolution 1 into type I wells, position 2 to load solution 2 into type IIwells, position 3 in which all wells overlapped with thermal expansionwells, and position 4 enabling solution 1 and solution 2 to mix witheach other.

Digital Immuno—PCR

Also disclosed is the application of digital immuno-PCR to measureproteins at single molecule level. As compared with a bulk system, thedigital format utilizes compartmentalization of single molecules into arelatively small volume, generating high local concentration. Secondly,compared with enzyme amplification, PCR amplification has severaladvantages: higher sensitivity, higher amplification efficiency, and nodead time. Anti-PSA capture antibody coated fluorescent magnetic beads(red) were used to capture the target PSA molecule.

The concentration of PSA was controlled so there was less than onemolecule on one bead. A dsDNA tag was attached to an anti-PSA detectionantibody and used as signal probe. After incubation between antibodiesand antigen, magnetic beads with captured/labeled PSA were loaded intopL wells with PCR supermix. Each well contains either one or no bead.After amplification, only wells containing beads were counted. The ratiobetween “on” wells and the total number of wells were used to determinethe concentration of target. Results are shown in FIG. 20.

LAMP Amplification

Digital reverse transcription loop mediated isothermal amplification(RT-LAMP) can be performed on the disclosed devices. In someembodiments, digital RT-LAMP is performed on a multivolume SlipChip™device.

In one illustrative case, one-step digital RT-LAMP is carried out bymixing template, primers, detection reagent, reaction mix and enzyme,then loading the solution onto a SlipChip device and heating up thedevice to a proper temperature for a period of time.

In one suitable example, the following mixture of reagents was used: 20μL reaction mix, 2 μLenzyme mix (Loopamp RNA Amplification Kit fromEiken Chemical Co., LTD.), 2 μL detection reagent (Eiken Chemical Co.,LTD.), 2 μL 20 mg/mL BSA, 8 μL RNase free water, 4 uL primer mix and 2μL HIV RNA purified from AcroMetrix® HIV-1 Panel 1E6. The finalconcentration of primers was 2 μM for BIP/FIP, 1 μM for LOOP primers,0.25 μM for B3/F3. All solutions were operated on ice.

The solution was loaded onto a multivolume SlipChip device (devicedesign published in Shen, F.; Sun, B.; Kreutz, J. E.; Davydova, E. K.;Du, W. B.; Reddy, P. L.; Joseph, L. J.; Ismagilov, JACS 2011 133: 17705)and the relative position of the plates of the device were fixed by wax.The device was heated on a thermal cycler block (Eppendorf) at 63° C.for about 1 hour then terminated at 95° C. for 2 minutes. Thefluorescence image was acquired by Leica DMI 6000 B epi-fluorescencemicroscope with a 5×/0.15 NA objective and L5 filter at roomtemperature. The measured concentration of digital RT-LAMP was 10% ofthat from digital RT-PCR using B3/F3 as primers.

In another embodiment, two-step digital RT-LAMP is carried out in twoseparate steps. Reverse Transcription is done by mixing template,BIP/FIP primers, reverse transcriptase, and reaction mix in a tube, andheating to a proper temperature. RNase H can be added afterwards to helpthe dissociation of DNA: RNA hybrid. Digital LAMP is performed by mixingobtained cDNA solution with all other components, loading the solutiononto a SlipChip device, and heating the device at a proper temperaturefor a period of time.

In another embodiment, digital RT-LAMP is performed by running thereverse transcription step on the SlipChip device in a digital format,mixing the product with other components of LAMP on-chip and heating thedevice. The result of this protocol has been experimentally observed tobe the same as when performing the RT step in a test tube.

Other embodiments that effect reactions (e.g., LAMP) operate viaprocesses that include compartmentalizing materials, effecting a firstreaction, and then effecting a second reaction. In one such embodiment,digital RT-LAMP is carried out by sequential reactions on multiplecompartments. First compartments are created with the solutioncontaining molecule of interest, BIP/FIP primers, reverse transcriptase,and reaction mix. The first reaction of reverse transcription is carriedout at 50 degrees Celsius for 15 minutes to synthesize cDNA. The secondcompartments are created with LAMP reagents and combined with the firstcompartments to carry out the second reaction. This second reaction ofLAMP is carried out at 63 degrees Celsius for 1 hour.

In one embodiment, multiplex LAMP can be carried out by sequentialreactions on multiple compartments. primers can be patterned in reactionareas (wells or surfaces) of a SlipChip, for example, as described inAnalytical Chemistry 2010 82:4606-4612, or the primer solution can beuser-loaded as described in JACS 2010 132: 106-111. The LAMP reagentcontaining molecule of interest is compartmentalized and mixed withpreloaded primers and the first reaction of amplification is carriedout. The second compartments containing detection reagent are createdand combined with the first compartments to carry out the secondreaction of detection.

In one set of experiments performed with two-step digital RT-LAMP, 10 μLreaction mix, 1 μL 20 mg/mL BSA, 0.5 μL Superscript III reversetranscriptase (Invitrogen), 6 μL RNase free water, 0.5 uL BIP/FIP primermix (10 μM) and 2 μL HIV RNA purified from AcroMetrix® HIV-1 Panel 1E6were mixed together in a test tube. All solutions were operated on ice.The solution was heated to 50° C. for 15 min for reverse transcription.

All other components of LAMP mixture (2 μL enzyme mix, 2 μL detectionreagent, 10 μL reaction mix, 1 μL 20 mg/mL BSA, all other primers andRNase free water to make up the volume to 20 μL) were mixed togetherwith the solution obtained from reverse transcription and loaded on aSlipChip device immediately. The whole device was heated on a thermalcycler block (Eppendorf) at 63° C. for about 1 hour then terminated at95° C. for 2 minutes. Imaging settings were the same as described forthe one-step RT-LAMP experimental protocol above. The measuredconcentration obtained after performing digital RT-LAMP was found to be30% of that from digital RT-PCR using B3/F3 as primers.

In another set of experiments, the efficiency of two-step digitalRT-LAMP was found to be improved by adding only BIP/FIP primer in the RTstep, adding RNase H after the RT step and removing B3 from the primermixture.

For example, 10 μl reaction mix, 1 μL, 20 mg/mL BSA, 0.5 μL SuperscriptIII reverse transcriptase (Invitrogen), 6 μL RNase free water, 0.5 uLBIP/FIP primer mix (10 μM) and 2 μL HIV RNA purified from AcroMetrix®HIV-1 Panel 1E6 were mixed together. All solutions were operated on ice.The solution was heated to 50° C. for 15 min for reverse transcriptionthen followed by the addition of 0.5 μL RNase H (NEB) and incubation at37° C. for 10 minutes.

All other components of LAMP mixture (2 μL enzyme mix, 2 μL detectionreagent, 10 μL reaction mix, 1 μL, 20 mg/mL BSA, all other primersexcept for B3 and RNase free water to make up the volume to 20 μL) weremixed together with the solution obtained from reverse transcription andloaded on a SlipChip device immediately. Heating and imaging settingswere the same as described for the two-step RT-LAMP experimentalprotocol above. The measured concentration after performing digitalRT-LAMP was found to be 60% of that obtained via digital RT-PCR usingB3/F3 as primers.

In another set of experiments, the efficiency of two-step digitalRT-LAMP was found to be improved by adding only BIP/FIP primer in the RTstep, adding thermostable RNase H into the LAMP mixture and removing B3from the primer mixture. Herein two step RT-LAMP was performed entirelyon SlipChip device, where the product of first step RT was introduced tothe second step LAMP by the overlapping of two sets of wells.

For example, 10 μL reaction mix, 1 μL 20 mg/mL BSA, 0.5 μL SuperscriptIII reverse transcriptase (Invitrogen), 6 μL RNase free water, 0.5 uLBIP/FIP primer mix (10 μM) and 2 μL HIV RNA purified from AcroMetrix®HIV-1 Panel 1E6 were mixed and added onto SlipChip device. All othercomponents of LAMP mixture (24 enzyme mix, 24 detection reagent, 10 μLreaction mix, 1 μL 20 mg/mL BSA, all other primers except for B3 andRNase free water to make up the volume to 19.5 μL) and 0.5 uL Hybridase™Thermostable RNase H (Epicenter) were mixed together and loaded intoanother set of well on the same SlipChip device. The whole device washeated on a thermal cycler block (Eppendorf) at 50° C. for 15 minutesthen slipped to allow overlapping of two sets of wells. Then the devicewas heated at 63° C. for about 1 hour then terminated at 95° C. for 2minutes. The imaging settings were the same as described for thetwo-step RT-LAMP experimental protocols above. The measuredconcentration after performing digital RT-LAMP was found to be 60% ofthat obtained from digital RT-PCR using B3/F3 as primers.

To perform digital RT-LAMP, the primers we used have the followingsequences:

LOOP_B:  GAGAACCAAGGGGAAGTGA LOOP_F:  TTTAACATTTGCATGGCTGCTTGAT BIP: TAT TGC ACC AGG CCA GAT GAT TTT GTA CTA GTA GTT CCT GCT ATG FTP: CAG CTT CCT CAT TGA TGG TCT CTT TTA ACA CCA TGC TAA ACA CAG T F3: ATT ATC AGA AGG AGC CAC C B3:  CAT CCT ATT TGT TCC TGA AGG

They are modified based on a published paper.

-   Curtis, K. A.; Rudolph, D. L; Owen, M. Journal of Virological    Methods 151 (2008) 264

The design and principles of primers were referred to

-   http://loopamp.eiken.co.jp/e/lamp/primer.html

FIP: Forward Inner Primer (FIP) consists of the F2 region (at the 3′end) that is complementary to the F2c region, and the same sequence asthe F1c region at the 5′ end.

F3 Primer: Forward Outer Primer consists of the F3 region that iscomplementary to the F3c region.

BIP: Backward Inner Primer (BIP) consists of the B2 region (at the 3′end) that is complementary to the B2c region, and the same sequence asthe B1c region at the 5′ end.

B3 Primer: Backward Outer Primer consists of the B3 region that iscomplementary to the B3c region.

The Loop Primers (either Loop Primer B or Loop Primer F), containingsequences complementary to the single stranded loop region (eitherbetween the B1 and B2 regions, or between the F1 and F2 regions) on the5′ end of the dumbbell-like structure

Additional background may be found in the following documents, each ofwhich is incorporated herein by reference in its entirety for any andall purposes.

-   (1) Livak, K. J.; Schmittgen, T. D. Methods 2001, 25, 402-408.-   (2) Vet, J. A. M.; Majithia, A. R.; Marras, S. A. E.; Tyagi, S.;    Dube, S.; Poiesz, B. J.; Kramer, F. R. Proc. Natl. Acad. Sci.    U S. A. 1999, 96, 6394-6399.-   (3) Mackay, I. M.; Arden, K. E.; Nitsche, A. Nucleic Acids Res.    2002, 30, 1292-1305.-   (4) Jarvius, J.; Melin, J.; Goransson, J.; Stenberg, J.;    Fredriksson, S.; Gonzalez-Rey, C.; Bertilsson, S.; Nilsson, M. Nat.    Methods 2006, 3, 725-727.-   (5) Vogelstein, B.; Kinzler, K. W. Proc. Natl. Acad. Sci. U S. A.    1999, 96, 9236-9241.-   (6) Nacht, M.; Dracheva, T.; Gao, Y. H.; Fujii, T.; Chen, Y. D.;    Player, A.; Akmaev, V.; Cook, B.; Dufault, M.; Zhang, M.; Zhang, W.;    Guo, M. Z.; Curran, J.; Han, S.; Sidransky, D.; Buetow, K.;    Madden, S. L.; Jen, J. Proc. Natl. Acad. Sci. US. A. 2001, 98,    15203-15208.-   (7) Cheng, B.; Landay, A.; Miller, V. Curr. Opin. HIV AIDS 2008, 3,    495-503.-   (8) Preiser, W.; Drexler, J. F.; Drosten, C. PLoS Med. 2006, 3,    e538; author reply e550.-   (9) UNAIDS/WHO 2008 Report on the Global AIDS Epidemic, UNAIDS/WHO,    2008.-   (10) Fan, H. C.; Quake, S. R. Anal. Chern. 2007, 79, 7576-7579.-   (11) Lo, Y. M. D.; Lun, F. M. F.; Chan, K. C. A; Tsui, N. B. Y.;    Chong, K. C.; Lau, T. K.; Leung, T. Y.; Zee, B. C. Y.; Cantor, C.    R.; Chiu, R. W. K. Proc. Natl. Acad. Sci. U SA. 200˜104,    13116-13121.-   (12) Heid, C. A; Stevens, J.; Livak, K. J.; Williams, P. M. Genome    Res. 1996, 6, 986-994.-   (13) Gibson, U. E. M.; Heid, C. A; Williams, P. M. Genome Res. 1996,    6, 995-1001.-   (14) Kalinina, O.; Lebedeva, I.; Brown, J.; Silver, J. Nucleic Acids    Res. 1997, 25, 1999-2004.-   (15) Sykes, P. J.; Neoh, S. H.; Brisco, M. J.; Hughes, E.; Condon,    J.; Morley, A A Biotechniques 1992, 13, 444-449.-   (16) Beer, N. R.; Wheeler, E. K.; Lee-Houghton, L.; Watkins, N.;    Nasarabadi, S.; Hebert, N.; Leung, P.; Arnold, D. W.; Bailey, C. G.;    Colston, B. W. Anal. Chern. 2008, 80, 1854-1858.-   (17) Kiss, M. M.; Ortoleva-Donnelly, L.; Beer, N. R.; Warner, J.;    Bailey, C. G.; Colston, B. W.; Rothberg, J. M.; Link, D. R.;    Leamon, J. H. Anal. Chern. 2008, 80, 8975-8981.-   (18) Leng, X. F.; Zhang, W. H.; Wang, C. M.; Cui, L. A; Yang, C. J.    Lab Chip 2010, 10, 2841-2843.-   (19) Ottesen, E. A; Hong, J. W.; Quake, S. R.; Leadbetter, J. R.    Science 2006, 314, 1464-1467.-   (20) Sundberg, S. O.; Wittwer, C. T.; Gao, C.; Gale, B. K. Anal.    Chern. 2010, 82, 1546-1550.-   (21) Applied Biosystems, Life Technologies. TaqManÂ OpenArrayÂ    Digital PCR Plates, 2010    https:1/products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catN    avigate2&catiD=607965.-   (22) Shen, F.; Du, W. B.; Kreutz, J. E.; Fok, A; Ismagilov, R. F.    Lab Chip 2010, 10, 2666-2672.-   (23) Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe,    K.; Amino, N.; Hase, T. Nucleic Acids Res. 2000, 28-   (24) Compton, J. Nature 1991, 350, 91-92.-   (25) Piepenburg, O.; Williams, C. H.; Stemple, D. L.; Armes, N. A    PLoS Bioi. 2006, 4, 1115-1121.-   (26) Lizardi, P. M.; Huang, X H.; Zhu, Z. R.; Bray-Ward, P.;    Thomas, D. C.; Ward, D. C. Nature Genet. 1998, 19, 225-232.-   (27) Vincent, M.; Xu, Y.; Kong, H. M. EMBO Rep. 2004, 5, 795-800.-   (28) Hill, C.; Bott, M.; Clark, K.; Jonas, V. Clin. Chem. 1995, 41,    S107-S107.-   (29) Chelliserrykattil, J.; Nelson, N. C.; Lyakhov, D.; Carlson, J.;    Phelps, S. S.; Kaminsky, M. B.; Gordon, P.; Hashima, S.; Ngo, T.;    Blazie, S.; Brentano, S. J Mol. Diagn. 2009, 11, 680-680.-   (30) Dean, F. B.; Hosono, S.; Fang, L. H.; Wu, X H.; Faruqi, A F.;    Bray-Ward, P.; Sun, Z. Y.; Zong, Q. L.; Du, Y. F.; Du, J.; Driscoll,    M.; Song, W. M.; Kingsmore, S. F.; Egholm, M.; Lasken, R. S. Proc.    Natl. Acad. Sci. US. A. 2002, 99, 5261-5266.-   (31) Walker, G. T.; Fraiser, M. S.; Schram, J. L.; Little, M. C.;    Nadeau, J. G.; Malinowski, D. P. Nucleic Acids Res 1992, 20,    1691-1696.-   (32) Hellyer, T. J.; Nadeau, J. G. Expert Rev. Mol. Diagn. 2004, 4,    251-261.-   (33) Mazutis, L.; Araghi, A F.; Miller, 0. J.; Baret, J. C.; Frenz,    L.; Janoshazi, A; Taly, V.; Miller, B. J.; Hutchison, J. B.; Link,    D.; Griffiths, A D.; Ryckelynck, M. Anal. Chern. 2009, 81,    4813-4821.-   (34) Blainey, P. C.; Quake, S. R. Nucleic Acids Res. 2011, 39, e19.-   (35) Fang, X E.; Liu, Y. Y.; Kong, J. L.; Jiang, X Y. Anal. Chem.    2010, 82, 3002-3006.-   (36) Dimov, I. K.; Garcia-Cordero, J. L.; O'Grady, J.; Poulsen, C.    R.; Viguier, C.; Kent, L.; Daly, P.; Lincoln, B.; Maher, M.;    O'Kennedy, R.; Smith, T. J.; Ricco, A J.; Lee, L. P. Lab Chip 2008,    8, 2071-2078.-   (37) Esch, M. B.; Locascio, L. E.; Tarlov, M. J.; Durst, R. A Anal.    Chern. 2001, 73, 2952-2958.-   (38) Lutz, S.; Weber, P.; Focke, M.; Faltin, B.; Hoffmann, J.;    Muller, C.; Mark, D.; Roth, G.; Munday, P.; Armes, N.; Piepenburg,    O.; Zengerle, R.; von Steffen, F. Lab Chip 2010, 10, 887-893.-   (39) Birch, D. E.; Laird, W. J.; Zoccoli, A 1998. Nucleic acid    amplification using a reversibly inactivated thermostable enzyme.    U.S. Pat. No. 5,773,258 (30 Jun. 1998)-   (40) Liu, J.; Hansen, C.; Quake, S. R. Anal. Chern. 2003, 75,    4718-4723.-   (41) Thorsen, T.; Maerkl, S. J.; Quake, S. R. Science 2002, 298,    580-584.-   (42) Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chern.-Int.    Edit. 2003, 42, 768-772.-   (43) Tewhey, R.; Warner, J. B.; Nakano, M.; Libby, B.; Medkova, M.;    David, P. H.; Kotsopoulos, S. K.; Samuels, M. L.; Hutchison, J. B.;    Larson, J. W.; Topol, E. J.; Weiner, M. P.; Harismendy, O.; Olson,    J.; Link, D. R.; Frazer, K. A Nat. Biotechnol. 2009, 27, 1025-1031.-   (44) Li, L.; Boedicker, J. Q.; Ismagilov, R. F. Anal. Chern. 2007,    79, 2756-2761.-   (45) Zheng, B.; Ismagilov, R. F. Angew. Chern. Int. Ed. 2005, 44,    2520-2523.-   (46) Brouzes, E.; Medkova, M.; Savenelli, N.; Marran, D.;    Twardowski, M.; Hutchison, J. B.; Rothberg, J. M.; Link, D. R.;    Perrimon, N.; Samuels, M. L. Proc. Natl. Acad. Sci. US. A. 2009,    106, 14195-14200.-   (47) Du, W. B.; Li, L.; Nichols, K. P.; Ismagilov, R. F. Lab Chip    2009, 9, 2286-2292.-   (48) Liu, W. S.; Chen, D. L.; Du, W. B.; Nichols, K. P.;    Ismagilov, R. F. Anal. Chern. 2010, 82, 3276-3282.-   (49) Li, L.; Du, W.; Ismagilov, R. F. J Am. Chern. Soc. 2009, 132,    112-119.-   (50) Li, L.; Ismagilov, R. F. Annu. Rev. Biophys. 2010, 39, 139-158.-   (51) Shen, F.; Du, W. B.; Davydova, E. K.; Karymov, M. A; Pandey,    J.; Ismagilov, R. F. Anal. Chern. 2010, 82, 4606-4612.-   (52) Li, L. A; Karymov, M. A; Nichols, K. P.; Ismagilov, R. F.    Langmuir 2010, 26, 12465-12471.-   (53) Shamoo, Y.; Friedman, A M.; Parsons, M. R.; Konigsberg, W. H.;    Steitz, T. A Nature 1995, 376, 362-366.-   (54) Piche, C.; Schemthaner, J. P. J Biomol. Tech. 2005, 16, 239-247-   (55) Du, W. B.; Li, L.; Nichols, K. P.; Ismagilov, R. F. Lab Chip    2009, 9, 2286-2292.-   (56) Li, L. A; Karymov, M. A; Nichols, K. P.; Ismagilov, R. F.    Langmuir 2010, 26, 12465-12471.

What is claimed is:
 1. A method of amplifying a nucleic acid molecule,comprising: contacting (a) a sample comprising at least one nucleic acidmolecule disposed at a plurality of first areas, with (b) at least onecomponent of an amplification reagent disposed in a plurality of secondareas, the contacting being effected by placing the first and secondareas into direct fluid communication with one another; and thecontacting comprises effecting relative motion between a substratecomprising the first area with a substrate comprising the second area;and exposing the area having the at least one nucleic acid molecule toconditions effective for amplification of the at least one nucleic acidmolecule.
 2. The method of claim 1, wherein the amplification isessentially isothermal.
 3. The method of claim 1, wherein at least twoof the plurality of first areas differ from one another in volume,wherein at least two of the plurality of second areas differ from oneanother in volume, wherein at least one first area differs in volumefrom at least one second area, or any combination thereof.
 4. The methodof claim 1, wherein the relative motion comprises rotation, lineartranslation, or both.
 5. The method of claim 1 wherein placing the firstand second areas into direct fluid communication with one anothercomprises removing a barrier between the first and second areas.
 6. Themethod of claim 1, wherein at least 10 first areas are placed intodirect fluid communication with at least 10 second areas essentiallysimultaneously.
 7. The method of claim 1, wherein at least one of thefirst and second areas has a volume of from 0.1 nL to about 1000 nL. 8.The method of claim 1, further comprising introducing the sample to theplurality of first areas, introducing the at least one component of theamplification reagent to the plurality of second areas, or both.
 9. Themethod of claim 8, wherein introducing the sample to the plurality offirst areas comprises exerting the sample through a conduit in fluidcommunication with the plurality of first areas.
 10. The method of claim9, wherein the conduit is formed in a substrate that comprises theplurality of second areas.
 11. The method of claim 8, whereinintroducing the sample to the plurality of second areas comprisesexerting the sample through a conduit in fluid communication with theplurality second areas.
 12. The method of claim 11, wherein the conduitis formed in a substrate that comprises the first area.
 13. The methodof claim 1, further comprising distributing the sample between theplurality of first areas and a first control area and wherein sample atthe first control area remains uncontacted with the at least onecomponent of the amplification reagent.
 14. A device, comprising: afirst substrate comprising a first population of areas, at least onearea of the first population of areas having at least one satellite areadisposed proximate to the at least one area, the at least one satellitearea being adapted to retain material from the at least one area; asecond substrate comprising a second plurality of areas, the first andsecond substrates being engageable with one another such that relativemotion between the first and second substrates places at least some ofthe first population of areas in register with at least some of thesecond population of areas so as to place the first and second areasinto fluid communication with one another.
 15. The device of claim 14,wherein the at least one satellite area is adapted to retain materialnot retained by a first and second area placed into fluid communicationwith one another.
 16. The device of claim 14, wherein at least one ofthe first or second substrates has a thickness in the range of fromabout 10 micrometers to about 5000 micrometers.
 17. The device of claim14, wherein the relative motion gives rise to at least one reactionregion defined by an area of the first population of areas in fluidcommunication with an area of the second population of areas, the atleast one reaction region having a volume in the range of from about 1pL to about 1 microL.
 18. A method of effecting amplification of atleast one nucleic acid target molecule, comprising: contacting (1) asample material disposed in a plurality of first areas, the samplematerial comprising a nucleic acid target, and at least one of the firstareas containing one molecule of the nucleic acid target, with (2) areactant material disposed in a plurality of second areas, thecontacting being effected by pairwise placement of at least some of thefirst areas and at least some of the second areas into direct fluidcommunication with one another, the contacting effecting amplificationof at least one nucleic acid target molecule.
 19. The method of claim18, wherein the sample material comprises a reagent.
 20. The method ofclaim 18, wherein the reactant material comprises an amplificationreagent, and wherein the method further comprises exposing the at leastone of the first areas containing one molecule of the nucleic acidtarget to conditions effective for amplification of the one nucleic acidtarget so as to give rise to an amplification product.